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Neuropathology of Post-Stroke Depression:
Possible Role of Inflammatory Molecules and Indoleamine 2,3-dioxygenase
by
Amy Wong
A thesis submitted in conformity with the requirements for
the degree of Master of Science (MSc.)
Graduate Department of Pharmacology and Toxicology, University of Toronto
© Copyright by Amy Wong 2010
ii
Neuropathology of Post-Stroke Depression: Possible Role of Inflammatory Molecules and
Indoleamine 2,3 dioxygenase
Amy Wong, MSc Candidate, 2010
Graduate Department of Pharmacology, University of Toronto
ABSTRACT
The study evaluated whether the activity of the indoleamine 2,3 dioxygenase (IDO)
enzyme is increased post-stroke and contributes to the development of post-stroke depression
(PSD) via tryptophan (TRP) depletion and neurotoxic kynurenine (KYN) metabolite production.
The activity of IDO was measured using the KYN/TRP ratio. Participants were assessed for
depression severity using the Center for Epidemiological Studies Depression Scale (CES-D).
Blood TRP, KYN, large neutral amino acids and cytokines were measured and compared.
Fifty-four (mean age=69.9±15.2, male=52.7%, mean NIHSS=7.3±4.6) patients within
28.9±40.3 days of stroke were separated into two groups: non-depressed (n=38, CES-
D=6.1±4.9) and those with significant depressive symptoms (n=16, CES-D=26.8±10.8). Higher
mean KYN/TRP ratios were demonstrated in stroke patients with depressive symptoms (non-
depressed=69.3±36.9 vs. depressive symptoms=78.3±42.0, F3,50=4.61, p=0.006) after
controlling for LNAA (p=0.026) and hypertension (p=0.039). As the KYN/TRP ratio reflects
decreased TRP and increased neurotoxic KYN metabolites, both mechanisms may play an
etiological role in PSD.
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ACKNOWLEDGEMENTS
I would like to thank Dr. Krista Lanctôt for her continued support, encouragement and
motivation throughout the course of my studies. Her loving maternal nature coupled with her
outstanding commitment to her work has made my experience as a graduate student an
unforgettable journey. She is a true inspiration to myself and others around me.
I would like to thank Dr. Nathan Herrmann for his continued support, encouragement
and enormous knowledge in the field of psychiatry. He possesses a genuine thirst for excellence
in medicine without limiting himself to any boundaries--a rare trait that I respect and admire
wholeheartedly.
Thank you to both Drs. Krista Lanctôt and Nathan Herrmann for the opportunity to
broaden my horizons in the field of neuropsyhopharmacology research. They have always
made themselves available to my needs and have been patient during my times of struggle.
Their dedication to their work and kindness to others is a great example to their pupils and
motivates me to continually strive for my passions, never ceasing to reach beyond my wildest
dreams or highest expectations.
Thank you to Abby Li, Allison Gold, Jaclyn Cappell, Karen Levy, Kelly Smart,
Mahwesh Saleem, Philip Francis, Robert Mitchell, and Walter Swardfager for their
friendship, blessings, and encouragements. I came in as the slightly crazy one, and I am leaving
as the extraordinarily insane one. I am certain that we will meet once again long after this is all
over—though hopefully not as one of your patients (not for my benefit, but for yours).
Finally, I am most indebted to my Father and my family who have showered me with
their love and support since before I was even born. You have sacrificed so much for me—
some without me knowing, I’m sure—and I am humbly honoured to call myself your daughter,
and your sister. You are my rock, my beacon, my guidepost to life; but most importantly, you
are my best friend. I would not have survived some of my toughest obstacles and fears had it not
been for your strength and belief in my capabilities to succeed. I love you, always.
The skills and lessons I have learned during the past two years are forever internalized
within me. It was at Sunnybrook that I discovered an amount courage I never even knew I
possessed; I am no longer afraid to take the risks I always talk about but never have been able to
realize. I know that in the future, after reaching what then seemed like an impossible dream, I
will look back to this day with both corners of my mouth slightly curled upwards, my eyes
gleaming with understanding, gratitude, and merriment.
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TABLE OF CONTENTS
ABSTRACT ..................................................................................................................... 1
1 INTRODUCTION ......................................................................................................... 1
1.1 Statement of the Problem ..................................................................................... 1
1.2 Purpose of the Study and Objectives .................................................................. 2
1.3 Statement of Research Hypotheses and Rationale for Hypotheses ..................... 3
1.3.1 Primary Hypothesis ......................................................................................... 3
1.3.2 Secondary Hypothesis .................................................................................... 4
1.4 Review of the Literature ........................................................................................ 5
1.4.1 Post-stroke Depression ................................................................................... 5
1.4.2 Inflammatory Processes and Stroke ............................................................. 18
1.4.3 Inflammatory Processes and Depression ...................................................... 21
1.4.4 Current Hypotheses on the Etiology of Post-Stroke Depression .................. 25
1.4.5 Neurological Consequences of Reduced Serotonin Synthesis and the Formation of Neurotoxic Metabolites ...................................................................... 29
1.4.6 Rationale for This Study ................................................................................ 38
2 METHODS ................................................................................................................. 43
2.1 Study Design ...................................................................................................... 43
2.2 Subjects .............................................................................................................. 44
2.2.1 Inclusion and Exclusion Criteria ................................................................... 44
2.2.2 Demographics and Medical History ............................................................... 45
2.3 Clinical Assessments .......................................................................................... 47
2.3.1 Depression Scales ........................................................................................ 47
2.3.2 Conition Scales ............................................................................................. 49
2.3.3 Stroke Severity and Functional Disability ...................................................... 50
2.3.4 Blood Draw .................................................................................................... 51
2.4 Laboratory Analyses ........................................................................................... 51
2.4.1 Cytokine Assays ............................................................................................ 51
2.4.2 KYN Assay .................................................................................................... 53
2.4.3 Tryptophan and LNAA Assays ...................................................................... 54
2.5 CT Scan Analysis ............................................................................................... 54
2.6 Statistical Analyses............................................................................................. 55
2.6.1 Preliminary Data Transformations ................................................................. 55
2.6.2 Planned Analyses ......................................................................................... 56
2.7 Sample Size Calculations ................................................................................... 58
2.8 Control of Confounders ...................................................................................... 59
3 RESULTS ................................................................................................................... 59
3.1 Demographic and Clinical Characteristics .......................................................... 61
3.2 Assay Results ..................................................................................................... 64
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3.3 Biological Correlates of Post-Stroke Depression ................................................ 65
3.3.1 Hypothesis I: Inflammatory Markers of Depression ...................................... 65
3.3.2 Hypothesis II: Kynurenine Activation and Depression ................................... 69
4 DISCUSSION ............................................................................................................ 72
4.1 The Role of IDO Activation in Post-stroke Depression ...................................... 72
4.2 The Role of Cytokines in Post-Stroke Depression .............................................. 76
4.3 The Role of Hypertension in Post-Stroke Depression ........................................ 81
4.4 Limitations .......................................................................................................... 86
4.5 Recommendations for Future Research ............................................................. 88
4.6 Clinical Implications ............................................................................................ 91
5 REFERENCES .......................................................................................................... 96
APPENDIX .................................................................................................................. 122
Appendix A: Post-stroke Depression Study Patient Consent Form ......................... 122
Appendix B: Sunnybrook REB Approval Letter ....................................................... 126
Appendix C: York Central REB Approval Letter ...................................................... 129
Appendix D: Baycrese REB Approval Letter ........................................................... 130
Appendix E: St. John's REB Approval Letter ........................................................... 131
Appendix F: Toronto Research Institute Approval Letter ......................................... 132
Appendix G: Study Roles ........................................................................................ 133
Appendix H: Presentations and Publications ........................................................... 134
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LIST OF TABLES
Table 1: Mean KYN/TRP Levels in Various Depression, Neurodegenerative Diseases, and Inflammatory Illnesses ............................................................................................ 40
Table 2: Cyotkine Assay Sensitivities ............................................................................ 53
Table 3: Participant Ineligibility ...................................................................................... 61
Table 4: Participant Demographics and Assessment Scores by Depression Group ..... 62
Table 5: Stroke Lesion Characteristics by Depression Group ....................................... 63
Table 6: Cerebrovascular Risk Factors and Concomitant Medication Use by Depression Group.......................................................................................................... 64
Table 7: Distribution of Hypertensive Patients on Anti-Hypertensive Medication ........... 69
Table 8: Measured and Imputed Cytokine Plasma Levels by Depression Group .......... 70
Table 9: Cytokine Detectability by Depression Group.................................................... 70
LIST OF FIGURES
Figure 1. The KYN Pathway: TRP Metabolism and its Products .................................. 35
Figure 2. Patient Recruitment ........................................................................................ 60
Figure 3. Mean KYN/TRP ratios in stroke patients with and without depressive symptoms ..................................................................................................................... 65
Figure 4. Box-plot of unadjusted KYN/TRP ratios in stroke patients with and without depression ..................................................................................................................... 66
Figure 5. Correlation between CES-D total scores and KYN/TRP ratio ......................... 66
Figure 6. Correlation between LNAA concentrations and KYN/TRP ratio ...................... 67
Figure 7. Mean CES-D Scores by Hypertension Group ................................................. 68
Figure 8. Correlation between unadjusted IL-6 concentrations and KYN/TRP ratio ...... 71
1
1. INTRODUCTION
1.1 Statement of the Problem
Stroke is the third leading cause of death in Canada, with more than 50,000 strokes
occurring annually1. This figure amounts to approximately one stroke for every 10 minutes of
time that has elapsed. About 30% of stroke victims do not survive the acute attack1. For the
survivors, rehabilitation involves addressing the physical, cognitive, psychological and
psychosocial impairments that are common post-stroke sequelae. One of the major
psychological changes observed in post-stroke patients is depression.
Depression is a significant health problem among the elderly population leading to
diminished quality of life2, impaired daily functioning
3-6, greater health service use
7, poorer
perceived health7 and increased mortality risk
8. With regards to stroke, depression occurs in
approximately one-third of all reported cases9-14
and is the strongest predictor of quality of life
(QoL)15-18
. Consequentially, post-stroke depression (PSD) patients have increased functional
disabilities19-23
, increased health service utilization24, 25
, increased cognitive impairment26
,
higher mortality rates27-31
even after controlling for other factors such as associated illnesses and
prior stroke27, 28
and poorer rehabilitation outcomes32-34
compared to non-PSD patients.
Moreover, stroke survivors are at elevated risk for clinically significant depressive symptoms
even two years after stroke onset35
.
Although antidepressant medications can be prescribed, the drugs are not efficacious in
all depressed survivors despite the availability of different formulas acting upon distinct
neurological pathways. Even when effective for depressive symptoms, post-stroke cognitive
impairments frequently persist36
. Additionally, response rates to antidepressant treatments are
inconsistent between studies partly due to variable definitions of response. The high prevalence
of depression post-stroke (about three times greater than the general population) combined with
2
the impact of these symptoms on antidepressant response highlights the urgency to discover
possible pharmacological targets for disease management. The generation of reliable data
regarding the neurological, physical and social correlates impacting PSD is a crucial factor in
the field of stroke recovery. Ultimately, a sound understanding of PSD etiology may lead to
novel therapeutic approaches that can propagate into successful management of this stroke
outcome.
1.2 Purpose of the Study and Objectives
PSD remains profoundly problematic due to its high prevalence, negative impact, and
lack of effective treatments. Several hypotheses regarding the etiology of PSD have circulated
since the early 1980s, beginning with Robinson’s lesion location theory37
. It was published that
stroke victims suffering from left anterior lesions were more prone to PSD than those of right or
posterior lesions. His hypothesis not only stimulated several debates, but also generated the
formation of subsequent hypotheses by other groups exploring other biological and psychosocial
components relevant to the disease. Despite such increasing interest in PSD research, no
conclusive data have been collected to date. The primary objective of this study was to assess
novel biological components that may contribute to the etiology PSD. In particular,
concentration changes of specific inflammatory markers and their ability to modulate tryptophan
(TRP) metabolism post-stroke was investigated as a possible cause of PSD. Tryptophan was of
special interest since it acts as a precursor to both the mood modulatory neurotransmitter
serotonin and neurotoxic kynurenine (KYN) metabolites.
KYN is formed by metabolism of TRP by indoleamine 2,3-dioxygenase (IDO);
therefore, an increase in IDO activity as stimulated by increases in pro-inflammatory cytokines
post-stroke is thought to initiate the development of PSD. Here, it is hypothesized that a
decrease in serotonin synthesis accompanied by an accumulation of neurotoxic KYN
3
metabolites act as a possible mechanism of the disease. This study measured the KYN/TRP ratio
as a marker of IDO activity in order to elucidate the hypothesis. Additionally, measurements of
pro- and anti-inflammatory cytokines were taken and analyzed to examine the relationship
between cytokines, IDO, and PSD. Finally, demographic and clinical factors were characterized
between depressed and non-depressed stroke patients in order to elucidate any markers that may
contribute to post-stroke inflammatory response mechanisms.
1.3 Statement of Research Hypotheses and Rationale for Hypotheses
1.3.1 Primary Hypothesis
Hypothesis 1: Increased IDO enzyme activity will be found in stroke patients experiencing
depressive symptoms compared to non-depressed stroke patients as evidenced by an increased
kynurenine/tryptophan (KYN/TRP) ratio.
Rationale: Initially, an increased KYN/TRP ratio was documented among depressed
immunotherapy patients38
, in post-partum depression39
, and in major depression40
. Most
recently, our group demonstrated that an elevated KYN/TRP ratio was associated with
depression among coronary artery disease patients, a group similar with respect to age and a
high prevalence of cerebrovascular risk factors41
. It is hypothesized that post-stroke stimulation
of IDO activity increases the production of KYN from its TRP precursor. Since TRP also acts
as a precursor to serotonin synthesis42
, decreased levels of serotonin are left available for
synapses between mood regulatory neurons. In addition, KYN can be metabolized into
neurotoxic metabolites43-45
that may cause further damages to areas of the brain responsible for
processing emotions. The effect of decreased serotonin synthesis accompanied by an escalation
of neuronal injuries is hypothesized to play a major role in the etiology of PSD. Thus, it is of
interest to measure IDO activity as a potential marker of disease progression. Since IDO has an
extremely short half life, an indirect measurement of enzyme activity (KYN/TRP ratio) will be
4
utilized instead. The KYN/TRP ratio has been used as a sensitive estimate of IDO activity and
cellular immunity both in vivo and in vitro since the late 1980s
46, 47.
1.3.2 Secondary Hypothesis
Hypothesis 2: Increased levels of pro-inflammatory cytokines (IL-1, IL-6, TNF-, IFN-) and
decreased levels of anti-inflammatory cytokines (IL-10) will be found in stroke patients
experiencing depressive symptoms compared to non-depressed stroke patients.
Rationale: Early pre-clinical studies have documented several cases of behavioural disturbances
in animals administered a variety of pro-inflammatory cytokines48, 49
. These behavioural
changes were collectively termed ―sickness behaviors‖ and are thought to mimic those of
depressive symptoms observed in humans50, 51
. More recent experimental and epidemiological
studies have reported higher levels of inflammatory markers within the nervous and circulatory
systems of depressed people52-58
. Marked increases in peripheral pro-inflammatory cytokines
have been documented in several post-stroke cases59-64
. By linking the two findings together,
the development of depression in immunocompromised patients is thought to be cytokine-
mediated. This theory has been supported by reported cases of depression after cytokine
immunotherapy65-68
where pro- and anti-inflammatory cytokines are thought to have opposite
effects. Although data regarding the role of pro- and anti-inflammatory cytokines remain
controversial, it has been hypothesized that a balance between both inflammatory markers is
crucial for maintaining mental fitness69-71
. According to the mechanism proposed for the
primary hypothesis, elevations in pro-inflammatory cytokines and reductions in anti-
inflammatory cytokines contribute to IDO activation and may contribute to PSD.
5
1.4 Review of the Literature
1.4.1 Post-stroke Depression
1.4.1.1 Prevalence
One systemic review of observational studies reported a 33% prevalence of PSD at any
time after stroke10
. Although this number concurs with most PSD studies, it is difficult to define
the true prevalence of PSD due to methodological (evaluation time, study setting,
inclusion/exclusion criteria) and diagnostic (different cut-off scores in rating scales, non-
consistent use of structured-clinical interviews, clinical findings only) variability present
amongst studies. PSD may also be over-diagnosed due to similar somatic symptoms produced
by other medical illnesses, or under-diagnosed particularly in patients with co-morbid cognitive
impairment. The correct attribution of somatic symptoms is important since it is relevant to
accurate scoring of depression severity scales, namely the Hamilton Depression Scale (HAM-
D), Montgomery Asberg Depression Rating Scale (MADRS) and Beck Depression Inventory
(BDI). Furthermore, the frequency of PSD may increase in prevalence over time since
symptoms may even begin to appear one year post-stroke irrespective of the level of handicap13,
72, 73. Conversely, some patients with early onset PSD (0-3 months post-stroke) are not
necessarily affected at one year follow-up74, 75
. Ultimately, quantification of PSD prevalence
proves to be challenging due to the dynamic nature of the disease in both its early and late
stages.
1.4.1.2 Risk Factors
Although the prevalence of PSD remains unpredictable, the disorder has been
documented to affect a high percentage of stroke victims (at least 30%). Thus, it is important to
investigate predictive demographic factors early after stroke onset in order to determine those at
highest risk for the disease. One study has shown that stroke severity, non-lacunar stroke
6
subtype and the melancholy index of the HAM-D (which includes depressed mood, feelings of
guilt, work inhibition, psychic anxiety and general somatic symptoms) were significant risk
factors for PSD at 3 months follow-up76
, and adjustment for stroke severity increased the
predictive value of the melancholy index at the same follow-up. Other studies have
demonstrated that prior personal or family history of major depression75
, poor social support74
,
greater impairment in activities of daily living (ADLs)74, 77, 78
, cognitive impairment79
and one or
more negative life events during the six months prior to stroke75
increases risk for PSD. Time
needed for recovery may also play an important role as it has been documented that those who
have not yet recovered from the disease at the one year mark have a high risk of developing
chronic depression74
. Despite these separate findings a more recent systematic review reported
that only the degree of physical disability, stroke severity and cognitive impairment are
consistently associated with the disease80
.
Because stroke often occurs in discrete lesion locations, researchers have examined the
neuroanatomical correlates of depression to identify regions that would put one at most risk of
PSD. Currently, literature surrounding lesion location remains controversial. There is some
evidence that PSD is accompanied by left anterior and left basal ganglia lesions and proximity
to the frontal pole has been positively correlated with depressive symptoms37, 81-85
. However,
these results have not been consistency replicated23, 86
and the reasons for these discrepancies
are not known. Thus, ready identification of patients at risk for PSD remains complicated.
Further studies are necessary to support, add to, or even contrast the current evidence available
before a sound pattern of PSD risk factors can be elucidated.
1.4.1.3 Demographics
To date, little is understood on the demographic determinants related to PSD onset.
Whether age increases the likelihood of developing PSD remains controversial. A couple of
7
studies support a correlative relationship between the two variables16, 87
while others report no
association between them9, 19, 88, 89
. With gender, there have been reports that women are more
likely to suffer from depression in the general population; however many studies have found
minimal to no effect between gender and PSD16, 22, 87, 89, 90
. Despite these negative findings, a
more recent systemic review evaluating sex differences in the prevalence of PSD reported the
disease to be slightly more common among women than men after combining post-stroke cases
ranging from less than two weeks to 15 years91
. The same factors explaining the greater
prevalence of depression in women among cardiac patients may also be applicable to post-
stroke cases, including elements of genetics, psychosocial inequities, issues related to recovery,
differential support and access to rehabilitation92
. Interestingly, women93, 94
and stroke patients
less than 59 years of age94
have been found to be more susceptible to post-stroke anxiety (PSA),
a disease that often manifests with PSD.
More recently, racial and ethnic disparities have been examined as potential
demographic correlates for PSD. In one study, white, non-Hispanic acute stroke patients from a
national cohort of Department of Veterans Affairs were more likely to be diagnosed with PSD
even after adjusting for sociodemographic and clinical characteristics95
. Another study
examining the relationship between ethnicity and risk for post-stroke mortality reported that
Non-Hispanic Black veterans with a history of stroke had a higher risk for mortality than did
Non-Hispanic White veterans96
. Because PSD patients have been shown to have a higher
mortality risk than non-depressed stroke patients27-31
racial and ethnic factors may be important
predictors in the development of the disease. At this time, it remains unclear whether racial and
ethnic backgrounds can be utilized as correlates for PSD.
Whether the stroke victim was an inpatient or an outpatient may be predictive of PSD. A
systematic review reported that inpatients with left anterior lesions were significantly more
8
likely to have MDD than outpatients97
. Note however, that this finding was only applicable to
the acute stroke period as no difference in mood was found between in- and outpatients during
the chronic stroke period.
1.4.1.4 Clinical Diagnosis of PSD
It is well-known that both major and minor depression are possible consequences of
stroke, though the true prevalence of each remains less certain. Major depression can be
accurately diagnosed in those with stroke98, 99
using the Diagnostic and Statistical Manual
Version of Mental Disorders, Fourth Edition (DSM-IV)100
. According to the manual, a patient
is diagnosed with major depression if at least five of the following nine symptoms are met:
depressed mood, anhedonia, appetite/weight change, insomnia or hypersomnia, psychomotor
agitation or retardation, fatigue or loss of energy, feelings of worthlessness or guilt, diminished
concentration, or recurrent thoughts of death. The five symptoms must include either depressed
mood or anhedonia and must persist for at least two weeks prior to evaluation. In contrast,
minor depression must only meet three of the previously mentioned nine symptoms with either
depressed mood or anhedonia listed as one of the three symptoms. Although studies have
documented major depression to occur in approximately 25%89, 98
of all stroke patients, the
occurrence of minor depression remains more elusive, with a reported prevalence ranging from
14-31%89, 98
.
Currently, there is no consensus on how to define "early" and "late" onset PSD.
However, Robinson demonstrated that 30% of stroke survivors who are non-depressed at initial
evaluation in an acute hospital setting will be diagnosed with depression 6 months later101
.
Following that criterion, one study examined the individual characteristics of early and late-
onset PSD, where early and late-onset were described as 3-6 months and 24 months post-stroke
respectively. It was found that the frequency of melancholic, vegetative, and psychological
9
symptoms and morning depression were significantly greater in patients with early-onset post-
stroke major or minor depression than in patients with late-onset major or minor depression102
.
Furthermore, early-onset post-stroke major depression was associated with larger lesion
volumes, and late-onset post-stroke major and minor depressions were associated with poorer
social functioning. Another study reported that early-onset PSD is characterized by anxiety,
loss of libido and feelings of guilt, whereas later-onset PSD is more frequently associated with
diurnal variation of mood and social isolations103
. Still, the prognostic differences and clinical
patterns between early-onset and late-onset PSD remain quite vague and are suspected to result
from differences in the biological or psychological processes acquired from the stroke.
Recently, diagnosis of PSD has shifted the emphasis from expressed mood and feelings
to the patient’s affect and behavior104
. This is especially true in cases where PSD is complicated
by significant neurological and functional impairment. Dominant hemispheric strokes are often
associated with aphasia making it difficult for patients to articulate thoughts and emotions of
sadness, hopelessness and guilt. On the other hand, patients with non-dominant hemisphere
stroke may still have intact language but be stricken with speech prosody, thus affecting their
ability to comprehend the importance voice tone and attitude expression in their speech (e.g. the
ability to imply sarcasm through tone of voice).
Patients with language impairments are not the only group of individuals who require
special attention. Several somatic and vegetative symptoms that are important clinical features
of PSD may be misattributed to a simple distaste for hospital environment. For example, weight
loss due to post-stroke dysphagia may be ascribed to a disliking for hospital food. Insomnia
may be noted as difficulty sleeping in a hospital setting rather than a symptom of depression. In
all cases, the opposite effect may also be true where patients are misdiagnosed with depression
due to misinterpretation of the aforementioned symptoms. Note, that vegetative symptoms are
10
significantly more common in depressed patients over the first two years post-stroke103
and that
certain somatic symptoms, specifically loss of appetite, psychomotor retardation and fatigue
have been found to be predictive of depression99
. Thus, it remains crucial for physicians be
attentive to such symptoms despite their unspecific nature.
1.4.1.5 Hypertension and the Vascular Hypothesis of Depression
Cerebral blood flow (CBF) and vasomotor reactivity are specific hemodynamic changes
that play a major role in stroke. Once CBF falls below threshold, delivery of vital nutrients
through the brain's microvasculature becomes compromised. This subsequently leads to
detrimental effects on neuronal functioning. If further decline in regional CBF occurs, cellular
mechanisms governing ionic equilibrium are also jeopardized105
. Furthermore, patients with
stroke or transient ischemic attacks have reduced vasomotor reactivity, indicating that cerebral
arterioles are unable to dilate in order to compensate for increased blood demand106
.
Hypertension is well-known to the public by its simplest definition--a condition
characterized by consistently elevated arterial blood pressure. However, hypertension entails
much more complicated hemodynamic changes than the previous definition describes. With
respect to blood vessels, it is characterized by two main factors: (1) the structural remodeling of
cerebral arteries via increasing wall thickness/lumen ratio107
and (2) impaired endothelium-
mediated vasodilatation as a result of collagen build-up108
. Ultimately, the effects of decreased
lumen size and decreased vessel distensibility may reduce both CBF and cerebrovascular
reactivity. Abnormal changes in regional CBF have been documented in the past where
hypertensive subjects had decreased CBF to the subcortical regions of the brain as well as to the
limbic and paralimbic structures109
. Thus, not only may hypertension create a vulnerability state
for the development of neurodegenerative disorders via oxidative stress mechanisms, but it may
also be relevant to the development of depression since limbic structures of the brain are widely
11
known to regulate emotions and behaviour. Indeed, both of the aforementioned hemodynamic
properties have previously been found to be impaired in subjects with depressive symptoms110,
111 where a reduction in blood flow velocity was especially prevalent in subjects suffering from
a DSM-IV depressive disorder110
. Furthermore cerebrovascular reactivity (CVR) is
significantly improved after antidepressant treatment at 21 months follow-up111
. Interestingly,
CVR has also been found to be significantly reduced in a group of depressed patients free of
vascular risk factors112
. This suggests that major depression may be the actual cause of CVR
function rather than its consequence. A combination of results from both studies suggests that
the link between hypertension and depression may be bidirectional with both leading to
increased risk for stroke.
Because depressive syndromes have been shown to be comorbid with cerebrovascular
lesions and cardiovascular risk factors, a vascular hypothesis to depression was introduced by
Alexopoulos and colleagues in the late 1990s. Here, it was stated that ―cerebrovascular disease
may predispose, precipitate, or perpetuate some geriatric depressive symptoms‖, and are
uniquely attributed to cases of late-onset depression113
. Further research has described the
symptomatology of the disease as distinct from non-vascular depression with more cognitive
impairment, prominent psychomotor retardation, apathy, lassitude and pronounced disability114
.
However, not all studies report a specific symptom profile of vascular depression115
.
Nevertheless, the vascular depression hypothesis remains a topic of interest as positive
associations between cardiovascular risk factors (i.e. hypertension114
, hyperlipidemia116
),
cerebro-117
and cardiovascular diseases118
, diabetes119
and depression have been documented in
the past.
In terms of size and location of the stroke lesion, Alexopoulos and his team postulated
that the neurocircuitry responsible for processing emotions can be affected by not only one large
12
infarct but by several macrovascular or microvascular lesions. Here, neuroimaging data
consisted of acute infarctions and white matter changes confined to three frontal-subcortical
loops of the brain that were later described to be key features of vascular depression. In support
of the multi-lesion hypothesis, one study found no relationship between focal vascular
pathology and PSD in post-mortem elderly subjects31
. This finding suggests that the vascular
burden from chronic accumulation of multiple lesions may be a crucial determinant to the
etiology of PSD rather than a single large infarct from stroke120
. Two years prior, Brodaty and
colleagues proposed the same neuroanatomical change of focus for PSD stating ―depression
after stroke may be related to cumulative vascular brain pathology rather than side and severity
of single stroke‖121
.
1.4.1.6 Treatment of PSD
Currently, antidepressants are prescribed to either prevent the occurrence of PSD or as
treatment of a newly diagnosed case. In 2005, a Cochrane review was published examining the
efficacies of both indications for therapy within a post-stroke population122
. The results,
however, were discouraging as no evidence of improved mood or cognitive function was found
in either the treatment or prevention group. Even at best, there were only some documented
effects of reduced depression severity within the treatment group. These negative findings may
reflect heterogeneity between studies, including differences in patient characteristics, methods
of diagnosis, assessment tools of depression and cut-off scores used in those forms of
assessments. Furthermore, patient recruitment varied from a few days to two years after stroke
onset. This likely complicated the analysis since the balance of risks and benefits of
pharmacotherapy may change over various stages of stroke recovery. Finally, the primary goals
of therapy in many trials were unclear which made it difficult to define important outcomes such
as remission and recovery. As a result of the methodological limitations encountered during the
13
review process, no successful recommendations regarding the applicability of antidepressants to
post-stroke conditions were made.
In contrast to the Cochrane review, more recent data supports the use of antidepressants
for the prevention of the disease. One RCT examining the SSRI escitalopram found the drug to
be significantly superior to placebo in preventing the development of PSD123
. The same study
also reported that problem-solving therapy, a manual-based intervention that originated from
England for treating medical patients with depression124
, was significantly superior to placebo.
Other classes of antidepressants have also been effective in preventing PSD, though the study
designs were less rigorous. In one study, 5.7% of patients receiving the tetracyclic acid (TeCA)
mirtazapine developed PSD compared to 40% of non-treated patients125
. While those
proportions were significantly different, the study was not placebo controlled and participants
were not blinded to treatment, which likely introduced a bias in favour of mirtazapine. As well,
more than 90% of people screened for recruitment were not enrolled as participants.
Milnacipran, which can be classified as a serotonin and norepinephrine reuptake inhibitor
(SNRI), was given open label to 11 patients and compared to historical controls and found to be
associated with improved PSD in rehabilitative in-patients126
. Finally, a 2007 meta-analysis
exploring prophylactic uses of antidepressants significantly favoured the treatment group
outcome. The study examined 10 randomized clinical trials where a total of 703 non-depressed
post-stroke patients were identified and reported a difference in PSD occurrence rate of about
17% between treated and non-treated groups (PSD occurrence of 29.17% and 12.54% in non-
treated and treated groups, respectively)127
. The authors also noted that the prophylactic effects
of antidepressants were not related to duration of use. However, the previously quoted studies
were the basis of that meta-analysis, thus study quality was a problem.
14
Additional publications suggest that antidepressants may reduce other post-stroke
sequelae. Several studies have suggested their positive effect on abating symptoms of cognitive
impairment that are often found co-morbid with PSD128-130
, and these improvements are likely to
remain stable over the next 2 years in the absence of subsequent reinjury to the central nervous
system131
. It has been suggested that post-stroke recovery from impaired activities of daily
living (ADLs)132
and cognition133
, and reductions in aggression and irritability134
may also be
enhanced by antidepressants if treatment were to begin within the first few months of stroke
onset. Some physicians opt to prescribe antidepressants to stroke patients before a formal
diagnosis of depression is made in order to prevent such disabilities. Although the prophylactic
use of these drugs has been reported to significantly reduce the occurrence of PSD127
, it does not
completely eradicate the disease and may manifest into different disease characteristics of PSD
compared to patients without treatment. For example, one study randomly assigned non-
depressed patients to receive nortriptyline, fluoxetine, or placebo for three months and examined
the occurrence of depression thereafter135
. In patients treated with antidepressants, lesion
volume and degree of social impairment were associated with late-onset PSD. In contrast, those
who were on placebo developed PSD associated with the severity of ADL impairments. The
differences in the clinical and pathological correlates between groups suggest that the
pathophysiology of the disease changes with the use of antidepressants, and that these subtle
differences may be important to post-stroke recovery.
Physicians are often reluctant to prescribe antidepressants to older stroke patients co-
morbid for other medical illnesses due to the perceived risk of side effects (e.g. drug-drug
interactions) leading to morbidity. Therefore, the selection of the antidepressant class with the
lowest risk/benefit ratio is of great importance. Both selective serotonin reuptake inhibitors
(SSRIs)136-138
and tricyclic antidepressants (TCAs)139
have been suggested to significantly
15
reduce depressive symptoms in some PSD patients. However, the use of TCAs in elderly
populations with vascular disease remains a safety concern among the medical community. A
systematic review comparing SSRIs and TCAs in patients vulnerable to stroke and
cardiovascular disease found that those receiving TCAs were at significantly greater odds of
developing non-serious cardiovascular adverse events (AEs) than those on SSRIs140
. These
non-serious events include the following: heart palpitations, chest pain, angina, arrhythmia,
hypertension and hypotension-syncope. Note, however, that serious cardiovascular AEs such as
death, cerebrovascular disease, heart failure, TIA, stroke and MI were not associated with the
use of TCA.
Interestingly, a couple of studies have documented potential benefits to SSRI use in
cardiovascular disease patients. Most notably, congestive heart failure (CHF) patients on active
aspirin medication yielded additional anti-platelet benefits during concomitant therapy with
SSRIs141
. Prior data from the same research group found that the SSRI sertraline and its
neurologically inactive metabolite N-desmethylsertraline (NDMS) exhibited potent, dose-
dependent antiplatelet activity in both in vitro
142 and ex vivo
143 clinical scenarios in patients
undergoing coronary stenting. These findings suggest that the use of SSRIs after ischemic
stroke may translate into improvement of depressive symptoms and a decreased rate of mortality
that would previously have been caused by unwanted cardiovascular AEs.
The findings above are likely the reason why SSRIs remain the most recommended
treatment for PSD due to their believed favourable tolerability (e.g. less serious cardiovascular
side effects, lack of anticholinergic effects)144, 145
. However, one must not discount the negative
side effects of SSRIs, as they are known to elicit sexual dysfunction, weight gain, and sleep
disturbances during long-term therapy146
. A recent study in post-menopausal women reported
that those using SSRIs had a 45% increased relative risk of incident stroke and 32% increased
16
risk of death compared to those with no antidepressant use
147. When analyzed by stroke type,
SSRI use was associated with incident hemorrhagic stroke and fatal stroke. The results are
consistent with prior evidence suggesting SSRIs may increase the risk of abnormal bleeding due
to their antiplatelet effects when combined with aspirin141
. Although the risk of death was
similarly increased among TCA users, no significant differences in risk between SSRI and TCA
users were found. However, it remains difficult to determine whether the non-significant results
can be attributed to drug-type exposure alone or unaccounted differences in other cardiovascular
risk factors. For example, depression has been an established risk factor for cardiovascular
morbidity and mortality148
and has been associated with an increased risk of stroke149
. This
raises the possibility that any ―excess‖ risk to stroke may be caused by depression and not solely
on drug use. Thus, it is necessary to assess the effect of different antidepressants specifically on
the PSD population in order to clarify their side effects. The optimal length of treatment is
unclear, though one research group recommends carrying on treatment for 4-6 months followed
by slow withdrawal of the drug150
. Conversely, one meta-analysis reported that the prophylactic
effects of antidepressants were not related to the duration of drug intake127
. Since there has been
several documented cases of robust placebo responses during antidepressant trials151
, it would
be necessary to examine the significance of placebo-effects before determining the best
treatment time-course for PSD patients.
1.4.1.7 Co-morbid Depression and Cognitive Decline Post-stroke
Cognitive symptoms are hypothesized to be indicators of chronic depression in the
elderly and have been found to persist in major depressive disorder (MDD) outpatients in
remission152-156
. Although the appearance of cognitive deficits remains under scrutiny, several
studies suggest that it may be immune-mediated. For example, higher levels of inflammation
during baseline assessments have been shown to predict cognitive symptoms at follow-up in
17
depressed patients157
. Thus, inflammation may act as an initiator and contributor to cognitive
symptoms of depression in the overall progression of the disease.
It has been reported that cognitive function and depressive symptoms are independently
associated with risk of mortality within the elderly population158
. Here, the study participants
were stratified into best, middle and worst groups based on scores collected from various
depression and cognition scales. It was found that older participants in the worst cognitive
function or worst depressive symptoms tertile at baseline had higher rates of mortality than
those in the middle and best tertiles. Furthermore, for each grouping of cognitive function,
greater depressive symptoms were associated with higher mortality rates, and for each grouping
of depressive symptoms, worse cognitive function was associated with higher mortality rates.
Thus, the combination of poor cognitive function and depressive symptoms appear to increase
the likelihood of mortality in an additive fashion, suggesting that the risk for poor outcome is
best explained by the cumulative effects of two or more risk factors.
With relation to post-stroke periods, one study related cognitive functioning to
depression and anxiety whereby verbal learning and reduced cognitive speed were correlated
with increased depression and anxiety scores159
. Furthermore, stroke patients with both
depression and cognitive impairment have been shown to present with longer durations of
depression than depressed patients without cognitive impairment160
. This finding was most
pronounced during the initial evaluation period of the study and highly associated with left
hemispheric lesions. Whether the acute post-stroke period and left hemispheric lesions are
absolutely related to co-morbid depression and cognitive decline requires further research.
However, it is apparent that cognition and mood post-stroke are linked and neither should be
ignored during the rehabilitative process.
_____________________________________
18
1.4.2 Inflammatory Processes and Stroke
1.4.2.1 Time course of inflammatory molecules post-stroke
Experiments inducing focal ischemia in rats via medial cerebral artery occlusion
(MCAO) have given insight to the inflammatory events that occur post-stroke. Circulating
monocytes have been detected within the capillaries and venules after 4 to 6 hours of induced
ischemia161
. The study also detected accumulation of polymorphonuclear neutrophils (PMNs)
12 hours after the same injury161
, though PMN infiltration may occur within 6 hours and with
greater severity in reperfused tissues than in permanently occluded tissues162
. Mechanistically,
it has been reported that leukoctyes and PMNs exacerbate tissue damage via edema formation,
physical obstruction through adherence to the vessels, and the release of oxygen radicals, pro-
inflammatory cytokines, and cytolytic enzymes leading to cellular necrosis162-164
. In support of
these findings, administration of inflammatory markers into rat models has been found to
augment increases in brain water content and tended to correlate with neutrophilic infiltration
into the parenchyma165
.
1.4.2.2 Cytokines
Cytokines are small, soluble glycoproteins (~15-25kd) that function locally or
systemically to orchestrate immune responses. They are produced by a variety of activated cell
types including endothelial cells, platelets, leukocytes, and fibroblasts166
. Additionally,
autocrine, paracrine, or endocrine effects are possible, depending on the particular cytokine105
.
Once released into the environment in response to an activating stimulus (e.g. stroke), they
control the growth and differentiation of different cell types through specific membrane
receptors105
. Ultimately, intracellular signaling pathways are activated and alter gene
transcription patterns within the cell, one of which modulates the expression of their own
specific target cell receptor.
19
In addition to being mediators of inflammation, cytokines also induce the expression of
cell adhesion molecules on endothelial cells that propagates cerebral ischemia and contribute to
tissue damage via alterations in levels of endogenous mediators such as tissue-plasminogen-
activator (tPa)105
. One post-mortem study of elderly patients showed that intercellular adhesion
molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) were increased in the
cerebral endothelium in late life depression167
. Combining these two findings suggests a
possible causal relationship between inflammatory cytokines and MDD.
1.4.2.3 Cytokines and Stroke: Pro- Versus Anti-inflammatory Cytokines
Cytokines are not stationary inflammatory markers and thus, are able to coordinate
immune responses between several physiological systems in the body. Post-stroke plasma
elevations of different cytokines168-172
(including IL-1, IL-6, IL-8, and TNF-) and their
mRNA expression in mononuclear cells168-172
have been previously reported and are
significantly correlated with brain infarct volume171, 173
, functional disability173
, risk of recurrent
stroke172
, and less favourable prognosis171
. Thus, not only is the inflammatory response related
to stroke severity, but clinical outcome as well. Two major categories of cytokines are the pro-
and anti-inflammatory cytokines which play opposite roles in regulating stroke outcome.
Pro-inflammatory cytokines (IL-1, TNF-α, IL-6, IL-8 and IL-18) function to amplify
immunological responses during reperfusion injury after acute cerebral ischemia. However,
they may also induce the depressogenic effects in depressed patients174, 175
. For example,
increased levels of IL-1β, TNF-α and IL-6 have been observed in the cerebrospinal fluid
(CSF)176, 177
and plasma178-183
of patients suffering from mood disorders. In addition, inhibition
of these cytokines and their respective signaling pathways can improve depressed mood184
.
Furthermore, both acute and chronic infectious and non-infectious diseases that primarily affect
immune regulation are often reported to be accompanied by depression185-188
. Note, however,
20
that the opposite is also true where immune dysregulation is found to be common in major
depression disorder, suggesting that depression by nature may be an autoimmune disease189
.
In contrast to pro-inflammatory cytokines, anti-inflammatory cytokines (IL-10, IL-4 and
insulin-like growth factors (IGF)) are proposed to have beneficial effect to the injured brain. In
one animal model, IL-10 administration to MCAO treated rats reduced infarct size compared to
vehicle treated controls190
. The opposite is also true where low levels of IL-10 have been linked
to larger infarct size191
and early neurological deterioration192
. Additionally, both IL-10 and
IGF have been shown to block lipopolysaccharide (LPS) and TNF- induced behavioural
changes in rats193, 194
. Although these results implicate neuroprotective properties of anti-
inflammatory cytokines to depression, their exact roles as immune modulators are clouded by
studies that have associated both increased192
and decreased195
IL-10 levels with poor outcome
and neurological worsening. Still, the balance between pro- and anti-inflammatory cytokines
must also be considered, highlighted by an elevated IL-6/IL-10 ratio in major depression69
.
Although the unfortunate prognosis of PSD has been well-documented, there is limited
research available explaining why certain post-stroke fates occur and the underlying
mechanisms involved in their development. With regards to mortality, the relationship between
pro- and anti-inflammatory cytokines may provide clues as to why the rate of mortality is
significantly higher in the PSD population compared to healthy aging individuals. Here, robust
evidence has been gathered from experiments supporting the development of an
―immunodepression syndrome‖, as summarized by two recent publications196, 197
. It is
hypothesized that the fate of injured brain tissue is dependent on the intricate balance between
pro- and anti-inflammatory cytokines where divergence from their naturally occurring
concentrations is thought to induce an immune response. Such prominent changes in
21
inflammatory markers are believed to facilitate systemic infection, thus resulting in a higher risk
for mortality post-stroke.
_____________________________________
1.4.3 Inflammatory Processes and Depression
1.4.3.1 Sickness Behaviour
Sickness behaviour is a collective term describing a constellation of behavioural
disturbances in animal models that mimic the depressive symptoms of humans. These
characteristics are comprised of malaise, decreased motivation (fatigue, lethargy, adipsia and
anorexia), decreased pleasure (anhedonia), psychomotor retardation, affective and cognitive
changes, hyposomnia or hypersomnia, decreased physical and social activities, poor
concentration and confusion198-202
. Current research suggests that inflammatory response
mechanisms may play a role in such behavioural disturbances. For example, administration of
the bacterial endotoxin LPS or inflammatory cytokines in animals produces some of the
behavioural characteristics encompassed in sickness behaviour48, 49, 203, 204
. Furthermore, the
absence of pro-inflammatory genes may inhibit the appearance of these depressive
characteristics. In support of this, one study found that mice deficient for the IL-6 gene are less
likely to exhibit the depressiogenic effects induced by LPS administration compared to mice
that do express the IL-6 cytokine205
.
1.4.3.2 Animal models of stroke
Behavioural correlates of depressive-like symptoms in animal models mainly include: 1)
reduced preference for sucrose solution when both the sweetened liquid and water are made
available206, 207
and 2) reduced locomotor and exploratory activity198
. The former observation is
an indicator of anhedonia and the latter suggests changes in incentive and motivation. A recent
study demonstrated both these symptoms in a novel animal model of stroke where MCAO
22
treated rats exhibited decreased locomotor activity and reduced sucrose solution preference208
.
Moreover, the observed behavioural abnormalities were ameliorated after the administration of
citalopram. These results are congruent with another study that also reported reduced sucrose
intake in MCAO-treated rats compared to control rats. However, the researchers took a
different approach to restore sucrose consumption. Instead, they administered the IL-1 receptor
antagonist (IL-1Ra) to MCAO-treated rats and found that it produced the same alleviation of
behavioural disturbances as antidepressants in the previously mentioned study209
. Results from
both findings not only confirm the presence of depressive symptoms post-stroke, but also
suggest that the disease may be immune-mediated.
1.4.3.3 Clinical Cases of Immunotherapy or Immune Challenges
Clinically, cytokines have been shown to be effective in the treatment of various medical
conditions including hepatitis C, leukemia, multiple sclerosis, Kaposi’s sarcoma, melanoma,
myeloma, renal carcinoma and other forms of cancer. However, cytokine immunotherapy may
also lead to pro-inflammatory molecule release causing depression210-217
, and may even
exacerbate preexisting psychotic symptoms218
. For example, the therapeutic use of cytokines
(e.g. IL-2, IFN-a) in the treatment of malignancies and chronic viral infections is frequently
complicated by cognitive, emotional, and behavioural disturbances219
. Moreover, antidepressant
therapy can prevent or attenuate cytokine induced depression220-223
and may even possess a
cytokine-linked anti-inflammatory effects222, 224-228
. These findings have opened up new
avenues for alternative treatments to depression, since pro-inflammatory cytokine inhibitors and
anti-inflammatory drugs may be able to prevent or attenuate depressive symptoms in humans.
In cases of immune challenges, one study found that mild stimulation of the primary host
defense in humans (using a safe and well-tolerated dose of endotoxin) produces negative effects
on both emotional and cognitive function229
. Elevations in both anxiety and depression levels
23
have been reported in other human cases of immune challenges186, 230
with those of lower
socioeconomic status most affected231
. All together, these findings suggest that pro-
inflammatory cytokines may play a crucial role in the pathogenesis of mood disorders in
humans, with implications for novel cytokine targeting therapies in neuropsychopharmalogical
research.
1.4.3.4 Cytokines in Clinical Depression and Cognitive Decline
The involvement of cytokines in the etiology of psychiatric disorders has been
extensively reviewed in the past by various authors174, 175, 184, 232, 233
. Under physiological
conditions, cytokine concentrations remain low in the body but are up-regulated during various
disease states, particularly those involving inflammation. With regards to depression, both
experimental and epidemiological research has found that depressed people have higher levels
of inflammatory markers52-58, 234
. For example, one study examined the levels of inflammatory
cytokines in a community-based sample of already depressed elderly subjects and found that
persons with depressed mood had higher median plasma levels of IL-6 and TNF-α57
. These
results became more significant after adjustments for health and demographic variables,
suggesting that depressed mood is either causing or caused by systemic inflammation.
Clinical studies have associated depression with increased levels of systemic cytokines
and acute phase proteins52, 58, 235-238
although such results were not always reproducible239, 240
.
Note, however, that the complex balance between pro- and anti-inflammatory cytokines may
play a crucial role in the development of mood disturbances, highlighted by reports of elevated
IFN- to IL-470, 241
and IL-6 to IL-1069
ratios in major depression. This may explain why a few
studies did not find a positive correlation between pro-inflammatory cytokines and depression
since none of them explored the significance of this balance. In addition, one of the authors
based their concentrations on lipopolysaccharide (LPS) induced peripheral blood mononuclear
24
cell (PBMC) cytokine production, which may be drastically different from the natural biological
concentrations of interest.
Cytokines are believed to affect cognitive function via active transport into the CNS
through afferent neurons that centrally activate cognitive responses, or upon local release by
activated microglia causing neurodegeneration242
. As previously mentioned, the
neuropsychiatric effects of cytokine therapy on cancer and HIV patients encompasses cognitive
changes that may involve a decline in verbal memory, cognitive speed and executive function216,
243, 244. Other forms of neurological disease associated with cognitive impairment have been
well documented in the past. Most notably, the severity of dementia in Alzheimer’s disease
patients is often accompanied with higher levels of circulating cytokines than controls, including
IL-1245-248
, IL-6247, 249, 250
, IL-18251
, and TNF-248
, although conflicting reports exist251, 252
. This
is not a disease related phenomenon since healthy aging individuals who experience cognitive
decline also exhibit higher levels of inflammatory markers253-257
independent of demographic
status and social status, and may also be at higher risk for mortality258
. Although the mechanism
of cytokine-induced cognitive changes has yet to be elucidated, it has been suggested to involve
damages to the fronto-subcortical circuitry. For example, one study found that stroke lesions of
the frontal lobe were associated with amusia (the absence of music perception), and that the
disorder also entailed general cognitive deficits in working memory, learning, semantic fluency,
executive functioning, and visuospatial coordination259
. Therefore, post-stroke elevations in
cytokines may contribute to post-stroke cognitive deterioration alongside depression.
The precise mechanisms behind cytokines and their ability to induce radical changes in mood
states remain unanswered. Generally, it is believed that increased peripheral cytokine levels
enter the brain and alter neurotransmitter metabolism, neuroendorcrine function, and neural
plasticity mechanisms responsible for mood regulation184
. Additionally, cytokine-mediated
25
interactions between neurons and glial cells may contribute to cognitive impairment (especially
in cases of memory and learning) via alterations in cholinergic and dopaminergic pathways242,
260. The next step would be to determine the cytokine concentrations and their time of activation
post-stroke to create a more detailed definition of their pathological outcome. Nevertheless,
elevations in cytokine levels may be useful prognostic markers for PSD despite their detrimental
effect to neuronal functioning.
_____________________________________
1.4.4 Current Hypotheses on the Etiology of Post-Stroke Depression
The etiology of PSD is thought to be multifactorial involving both psychosocial
vulnerability and biological mechanisms. In addition, several scientists believe that location of
the infarct plays a crucial role in understanding the mechanisms behind the disease. Thus, three
major hypotheses currently exist in the etiology of PSD: lesion location, psychosocial factors,
and biological factors261
.
1.4.4.1 Lesion Location Hypothesis
Those who support the lesion location hypothesis believe that PSD is directly caused by
focal damage to brain regions involved in the mood regulatory system. An early pioneer of the
lesion location hypothesis is Robinson and his team who were the first to report that left-
hemisphere lesions located to the vicinity of the frontal pole were more frequently associated
with PSD than lesions elsewhere in the brain82
. Since then a couple of other studies have
reported the same phenomenon74, 159, 262, 263
. These findings were encouraging to the lesion
location hypothesis since they support theories regarding: 1) frontal structures in the regulation
of emotional behavior and 2) lateralized differentiation in the organization of emotion, where
the left side of the brain is more often activated than the right side during emotional
processing264, 265
. In support of this, a MRI study found that left lesions of the frontal-subcortical
26
circuits (i.e. pallidum and caudate) predisposed stroke patients to depression with size of infarcts
being larger in depressed patients12
. Furthermore, the severity of depression has been associated
with left frontal lobe damage, with the exception of the basal ganglia266
. However, two more
recent systematic reviews267, 268
and several other studies76, 89, 269-273
failed to confirm the
association between left-sided strokes and depression, while one review stated such an
association varied depending on whether patients were sampled as inpatients or from the
community97
. Post-stroke cognitive impairment may also be related to lesion location as
comorbid MDD and cognitive impairment has been associated with left-sided lesions274
.
To counter the unsuccessful replication of their findings, Robinson and colleagues
carried out a meta-analysis275
and claimed to have found an interaction between lesion location
and time of PSD onset. Here, left-hemisphere lesions would increase the risk of PSD especially
in the first couple of months after stroke, whereas in the subacute to chronic course,
psychosocial factors would play a more important role. In addition, it was suggested that the
proximity of the left hemispheric lesion to the frontal pole was correlated with depression
severity, but not in cases of right-hemisphere stroke276
. However, prior to such claims, Gainotti
and his team had tested this same assumption and found that symptom profiles and the
anatomical and clinical correlates of major PSD were no different in the acute and chronic
stages of stroke270
. Currently, confirmation or rejection of Robinson and colleagues’ findings
has yet to be elucidated. However, many researchers agree that the etiology of PSD does not
and cannot rely on the lesion location theory alone.
1.4.4.2 Psychosocial Factors Hypothesis
The psychosocial hypothesis states that PSD is a psychological reaction to the physical
devastation caused by stroke whereby disability is the major predictor to disease development.
Several studies have supported this hypothesis22, 270, 277, 278
even after controlling for other stroke
27
characteristics including size and location of infarct85, 89, 279
. Lack of social companionship272, 273,
280 and absence of family support
74 have also been associated with the development of PSD and
are predictive of depression severity at six months post-stroke272
. Additionally, the development
of functional disabilities as a result of the stroke may lead to greater depressive symptoms which
would further complicate functional outcome20, 33, 281
. In support of this, improvement of
depressive symptoms post-stroke has been also associated with better functional recovery88
.
Although the severity of functional disability has been shown to be a strong predictor of
depression at 3 months post-stroke74, 85
the association disappears after around 12 to 24 months
post-stroke74, 85, 279
. Instead, few social contacts outside the immediate family and diagnosis of
the disorder at an in-hospital setting contributes most to depression at those times. Thus,
disability may only be a risk factor for depression in the acute and subacute post-stroke period,
where it does not determine the onset of depression per se, but interacts with it to limit the
results of long-term rehabilitation280
. These suggestive findings have led to the hypothesis that
PSD is at least, in part, a consequence of the direct biological consequence (e.g. immune
response) of the stroke.
1.4.4.3 Other Biological Factors
It has been hypothesized that during acute brain infarction, there is decreased
monoamine synthesis leading to decreased serotonin levels in the brain. In blood, serotonin is
present in high concentrations in platelets and released into the plasma when platelets aggregate
at the site of tissue damage. It is then subsequently catabolized by monoamine oxidase enzyme
activity in the liver and lungs. In the CNS, serotonin is present in high concentrations in
localized regions of the midbrain, serving as a neurotransmitter. The level of CSF or brain
serotonin remains controversial—some studies associate MDD with reduced levels of CSF
serotonin and increased levels of serotonin turnover282, 283
whereas other studies find no
28
differences in CSF concentrations of serotonin metabolite (5-hydroxyindoleacetic acid) between
depressed and healthy subjects284, 285
.
It has been proposed that PSD is caused by ischemic brain lesions that directly disrupt
neural circuits involved in mood regulation286
. In particular, it has been postulated that stroke
lesion interrupts the biogenic amine containing axons ascending from the brainstem to the
cerebral cortex, thereby leading to a decreased production of serotonin and norepinephrine in
uninjured limbic structures of frontal and temporal lobes as well as basal ganglia287
. Thus,
deficits in important mood regulatory neurotransmitters or failure of the body to up-regulate
their respective receptors after stroke may trigger PSD. This hypothesis has been supported by
studies demonstrating significantly lower CSF and plasma concentrations of serotonin288
and
CSF concentrations of its metabolite, 5-hydroxy-indoleacetic acid289
, in PSD patients compared
to non-depressed stroke survivors. Moreover, one study reported an inverse correlation between
serotonin receptor binding in the left temporal cortex and HAM-D scores of depressed
patients290
.
In further support of the biological hypothesis, one study found that the most commonly
reported symptoms in a group of post-stroke patients were discouragement/hopelessness, self-
criticalness, tiredness and feelings of being punished159
, all of which describe non-physical
symptoms. Furthermore, a community-based post-stroke cohort was found to have an elevated
risk for clinically significant depressive symptoms that was not mediated by functional
disability35
. Lastly, one study reported an association between the presence of small silent
strokes and late-onset depression, suggesting that patients who are unaware of their existing
cerebral lesions develop depression independently of psychological mechanisms related to
functional debilitation291
.
_____________________________________
29
1.4.5 Neurological Consequences of Reduced Serotonin Synthesis and the Formation of Neurotoxic Metabolites 1.4.5.1 Serotonergic Hypothesis of Depression
The serotonergic hypothesis of depression has been in existence for over 40 years292
and
proposes that diminished activity of 5-HT pathways is responsible for the pathopysiology of
depression. The hypothesis was evidenced by countless observations of patient response to
treatment with tricyclic antidepressants in which inhibition of 5-HT and noradrenaline reuptake
was presumed to enhance 5-HT-mediated neuroendrocrine responses. Moreover, the
development of the more efficacious SSRIs suggested that depressive symptoms can sufficiently
be attenuated as a result of increased synaptic 5-HT concentration293
. Today, 5-HT is
recognized to influence many symptoms of depression, including mood, appetite and sleep
patterns, physical activity, sexual interest and cognitive dysfunction.
Imaging investigations of the 5-HT receptors via ligand detection in conjunction with
positron emission tomography (PET) or single photon emission computed tomography (SPECT)
have provided consistent and convincing evidence that the binding density of 5-HT1A294, 295
and
5-HT2296
receptors is significantly decreased in MDD patients. The reduction in receptor
binding density was particularly prominent in areas of the brain involving the amygdala297
,
midbrain297, 298
, and brainstem299
. Age-related effects may also be of significant importance in
which reduced receptor binding density occurs with increasing age298, 300, 301
. Similar results
have been reported for the serotonin transporter (SERT), where drug-free unipolar depressed
patients exhibited reduced SERT availability in the brain stem299
. However, these findings are
not necessarily reproducible as no difference in SERT availability between depressed and
healthy volunteers has also been documented302
. Similarly, no significant difference in 5-HT
binding potential was found between recovered depressed patients and healthy controls in one
study303
while another study documented widespread persistent reductions in 5-HT receptor
30
binding among patients who have already recovered from the disease. Taken together, these
results suggest that reduced binding density may represent a genetic abnormality that confers
vulnerability to recurrent major depression rather than an observation made only during the
course of the disease303
.
In relation to post-stroke cases, the presence of an acute infarct is hypothesized to reduce
monoamine synthesis (e.g. 5-HT) within the brain. In the past, PSD patients have been
consistently documented to have lower CSF concentrations of the serotonin metabolite 5-
hydroxyindoleacetic acid compared non-PSD patients289
. More recently, one study measured
the CSF levels of serotonin in PSD patients288
and compared these levels to plasma
concentrations of serotonin. It was reported that serotonin concentrations in the CSF and
plasma of PSD patients were considerably lower than non-depressed patients and that a greater
number of PSD patients had lower than normal serotonin concentrations than the control group.
Furthermore, a sound correlation was found between the plasma and CSF serotonin
concentrations in both PSD and control patients, suggesting that plasma concentrations of
serotonin may be useful in predicting the central concentrations in patients with stroke or
depression.
1.4.5.2 Tryptophan and Central Serotonin Synthesis
Tryptophan is an essential amino acid that can only be obtained through external
sources. Once absorbed by the body, tryptophan travels around the peripheral circulation in
equilibrium between its albumin-bound and free form, the former comprising of 90% of the
body’s tryptophan level42
. Tryptophan can only be transported across the BBB in its free form
by the L-type amino acid transporter304
and once in the CNS, tryptophan acts as a precursor in
various metabolic pathways. The end products of these pathways are comprised of various
proteins, kynurenine, and serotonin42
. Note that brain TRP levels can be considered a reflection
31
of its plasma levels since TRP transport across the BBB directly affects the amount of
metabolites synthesized.
Tryptophan hydroxylase (TPH) is located in the cytoplasm of neurons of the raphe
nuclei and acts as the rate limiting enzyme of 5-HT synthesis. Using molecular oxygen and the
cofactor tetrahydobiopterin, it catalyzes the hydroxylation of TRP into 5-hydryoxytryptophan
(5-HTP). A subsequent decarboxylase enzyme then rapidly converts 5-HTP into 5-HT using
pyridoxal-5’-phosphate as co-factor42
. Serotonin is then either: 1) bound by the carrier protein
serotonin-binding protein (SBP) and released into the extracellular fluid (synapse) via calcium-
dependent exocytosis; or 2) metabolized into 5-hydroxyindoleacetic acid (5-HIAA) by
monoamine oxidase enzymes and excreted from the cell via energy dependent mechanisms.
Following release into the synapse, 5-HT can be recycled back into the neuron through SERT
reuptake located on the pre-synaptic membrane or metabolized into 5-HIAA in the post-synaptic
neuron.
Several researchers have examined the role of TRP and 5-HT in depression and the
clinical and neuropsychological consequences of abnormal 5-HT activity in the brain. For
example, one of the most reliable pieces of evidences linking 5-HT abnormalities to depression
was first documented in the mid-1980s, where total plasma tryptophan was significantly lower
in depressed patients compared to controls305
. More recently, researchers have constructed a
technique to induce a brief tryptophan depleted state in healthy subjects in order to assess the
mood effects of reducing central 5-HT function. This is simply accomplished by administering
an amino acid mixture free of TRP; thus, lowering both plasma and brain TRP concentrations.
Because the rate limiting step of 5-HT synthesis is determined by TPH activity, and because
TPH is only 50% saturated with TRP under normal physiological conditions306
, tryptophan
depletion is predicted to produce a transient lowering of central 5-HT activity.
32
The return of depressive symptomatology as a result of TRP depletion has been
documented in recovered depressed patients withdrawn from medication307, 308
. However, TRP
depletion appears insufficient to cause clinically defined depressive symptoms in healthy
volunteers308
. Although the difference in response to tryptophan depletion between the two
populations has yet to be elucidated, it has been postulated that depression may be the result of
persistent neurobiological changes that produce clinically significant mood abnormalities when
affected individuals are exposed to low 5-HT activity309
. The types of neurobiological changes
remain undefined but are hypothesized to involve altered or impaired innervations in
neurocircuitry that consistently leads to negative biases in emotional perception. Whatever the
mechanism, it can be said that tryptophan depletion studies generally support a casual
relationship between decreased central 5-HT and depression, and that those with risk factors for
depression or a family history of depression are most affected.
1.4.5.3 Central Tryptophan Availability
Knowledge underlying the specificities of tryptophan uptake into the serotonergic
neurons remains scarce. In its most simple definition, it is generally agreed upon that a
dynamic, regulatory barrier exists between plasma and intraneuronal tryptophan concentrations.
Although it is known that the L-type amino acid transporter is responsible for tryptophan
transport into the brain, two important factors surrounding this transport system must be
considered in central tryptophan availability: 1) the ratio between free tryptophan to its albumin-
bound form since only the free fraction crosses the BBB; and 2) the competitive nature of other
large neutral amino acids (LNAA = tyrosine, valine, leucine, isoleucine and phenylalanine) for
entry into the brain via the same transporter.
Several studies have reported lower tryptophan and tryptophan to LNAA ratio
(TRP/LNAA) in MDD patients compared to healthy controls305, 310-312
or subjects with obsessive
33
compulsive disorder (OCD)311
. The ratio appears to be most correlated with depressed mood313
,
retardation313
, and melancholic features314
. While some studies demonstrated reduction by using
total tryptophan, others have found reductions in only free tryptophan. Depression severity has
also been related to lower TRP/LNAA ratio among the depressed population315
, though the
opposite finding has also been documented312
. TRP/LNAA analysis may also successfully
predict treatment response to antidepressants, with a lower baseline TRP/LNAA ratio indicative
of greater improvements in depressive symptoms316-318
. Additionally, one study reported that
good responders exhibited a steady increase in TRP/LNAA ratio as the length of treatment
proceeded for six weeks319
. All in all, there is substantial evidence to support the relationship
between reduced central tryptophan availability and the presence of depression. Indeed,
increased brain tryptophan availability can increase brain 5HT synthesis320
. However, these
findings have yet to be tested among the PSD population.
1.4.5.4 Kynurenine Biosynthesis and Metabolism
Tryptophan is a ubiquitous molecule that is not only essential for protein synthesis but
plays a crucial role in central 5-HT metabolism. Additionally, the amino acid is a precursor for
the kynurenine pathway of metabolism in astrocytes, infiltrating macrophages, microglia and
dendritic cells321, 322
. The end products include kynurenine and several of its metabolites, some
of which are neurotoxic and others which are neuroprotectant. Under this pathway, the indole
ring of tryptophan is first metabolized by the rate limiting enzymes tryptophan 2,3-dioxygenase
(TDO), found highly concentrated in hepatic cells323
, or by indoleamine 2,3-dioxygenase (IDO),
most prominently expressed by cells in the CNS and infiltrating macrophages321, 324
. It can then
be inferred that TDO regulates homeostatic plasma tryptophan concentration in non-
inflammatory conditions while the extra-hepatic IDO predominantly maintains brain tryptophan
levels and is thus, of special interest in to PSD.
34
IDO is up-regulated in response to infection and tissue inflammation by certain
cytokines325-330
and can be induced in vitro by other inflammatory molecules such as LPS331
and
amyloid peptides332
. Once IDO metabolizes tryptophan into kynurenine (KYN) it proceeds
along the pathway until nicotinamide adenosine dinuleotide (NAD) is achieved as the final
product. Several neuroactive intermediates are generated during this process and comprising of:
1) 3-hydroxykynurenine333, 334
and 3-hydroxyanthranilic acid45
, both free-radical generators; 2)
quinolinic acid335
, the excitotoxin and selective N-methyl-D-aspartic acid (NMDA) receptor
agonist; and 3) kynurenic acid (KA)336
, a neuroprotective NMDA antagonist. A detailed
diagram of TRP metabolism and its products are displayed in Figure 1. Since the kynurenine
pathway produces both neurotoxic and neuroprotective metabolites, the ratio between these
products are of great importance when correlating their concentrations to the pathological
consequences of PSD. For example, the KA/KYN ratio has been show to be decreased in
depressed hepatitis C patients undergoing IFN- therapy337
and MDD patients40
.
Several pro-inflammatory cytokines possess the ability to induce IDO activity, including
IFN-338
, IFN-339
, IFN-338, 340
, IL-2341
, IL-6342
, IL-18343
, and TNF-331
. Among this list, IFN-
has consistently been reported to be the most potent regulator of the enzyme. Conversely, the
anti-inflammatory cytokine IL-4, IL-10 and TGf- act to inhibit IDO activity344
. Interestingly,
one study found that IL-10 is able to suppress IFN- induced IDO expression in cells derived
from the hypothalamic-pituitary-adrenal axis (HPA)329
. It cannot be stressed enough that the
balance between pro- and anti-inflammatory cytokines is crucial for homeostatic maintenance of
the human body. Therefore, an excess of pro-versus anti-inflammatory cytokines have the
potential to cause mood dysfunction in several neurological diseases if IDO activity exceeds its
normal boundaries of TRP metabolism.
35
Figure 1. The KYN Pathway: TRP Metabolism and its Products345
36
1.4.5.6 Linking Cytokines and Tryptophan Metabolism to the KYN/TRP ratio
A newer hypothesis to the etiology of depression involving the metabolic pathways of
tryptophan was recently described336
. Increased cytokine levels (such as in the case of stroke)
upregulates IDO activity, an enzyme that is widely distributed in the intestinal tissues, lungs,
placenta and brain291, 343, 346-349
. Because it is in direct competition with TPH for the metabolism
of tryptophan into 5-HT, heightened IDO activity as a result of elevated in cytokine levels may
reduce tryptophan availability for 5-HT production. Consequently, central serotonin
concentrations become sparse, leading to mood complications and behavioural disturbances in
the affected individual. In support of this hypothesis, several studies have shown a significant
decrease in serum TRP concentrations in patients treated with cytokines350-352
. In addition, TRP
levels have been found to be decreased in depressed patients314
while the administration of TRP
produces an antidepressant effect340
. Based on such findings, one is inclined to postulate that
clinical conditions associated with increases in pro-inflammatory cytokines lead to activation of
the IDO enzyme, subsequently resulting in decreased 5-HT synthesis and thus, increased risk of
depression.
We postulate that the mechanism above may have a significant effect on the etiology of
PSD. To reiterate, elevations in post-stroke cytokine levels shunts tryptophan metabolism from
the serotonin pathway into the kynurenine pathway via upregulation of the IDO enzyme. Under
the kynurenine pathway, tryptophan is catabolized into kynurenine; thus reducing the
availability of tryptophan to be converted into the mood regulatory neurotransmitter 5-HT.
Kynurenine is subsequently broken down into several neuroactive intermediates, including three
potent neurotoxins; the hydrogen peroxide generators, 3-hydroxykynurenine and 3-
hydroxyanthranilic acid223
and the NMDA agonist, quinolinic acid (QUIN)335
. Indeed, one
study reported IFN-α immunotherapy treatment in hepatitis C patients significantly increased
37
peripheral blood KYN353
. This was accompanied by marked increases in CSF KYN and QUIN
and correlated with the severity of depressive symptoms.
Several studies have examined the effects of the neurotoxic kynurenine metabolites. 3-
hydroxykynurenine levels are elevated in Huntington’s disease patients354, 355
and has been
shown to induce neuronal cell death in cortical and striatal cell cultures44
. Intracerebral
injection of 3-hydroxyanthranilic acid can decrease choline acetyltransferase activity356
, thus,
decreasing acetylcholine synthesis. QUIN is produced by infiltrating macrophages, microglia
and dendritic cells under inflammatory conditions and acts as a potent agonist of the neuronal
NMDA subtype of glutamate receptors335
. Moreover, several neurodegenerative conditions
have been linked to QUIN-induced apoptosis, including Huntington’s disease357
and HIV-
associated dementia358
. Most recently, it was suggested that KYN preferentially metabolized
along the QUIN pathway at the subcortical level (amygdala/striatum) in mice models359
. This
has its importance since a reduction in 5-HT receptor binding density has been associated with
MDD and is most pronounced in the amygdala. Altogether, it is possible that these metabolites
inflict further damage to the post-stroke brain by destroying essential neuronal pathways
responsible for regulating mood and cognition.
Because measurement of IDO activity has not been thoroughly explored in stroke and is
difficult to measure, the kynurenine to tryptophan ratio (KYN/TRP) has been proposed as a
marker for its activity instead. The KYN/TRP is a reliable marker of IDO activity based on its
reproducibility in past studies examining depression and related neurologic diseases. For
example, enhanced TRP degradation and higher KYN/TRP ratios in Alzheimer's disease,
Parkinson's disease, Huntington's disease, cancer, malaria, and rheumatoid arthritis have been
associated with advanced stages of the diseases and more severe symptoms and fatal outcomes
360-367. Furthermore, it has been reported that the inflammatory marker neopterin, is correlated
38
with both KYN/TRP ratio and kynurenine concentrations and inversely correlated with
tryptophan in cases of immune activation368, 369
. Elevations in the KYN/TRP ratio has not only
been associated with the severity of well-known inflammatory diseases but has also been linked
to cases of depression. Individuals undergoing cytokine therapy for hepatitis C and cancer
developed depressive symptoms that were specifically linked to increases in plasma KYN and
KYN/TRP ratio38, 337
. Similar increases in the KYN/TRP ratio have also been found in women
with post-partum depression39, 370
. Most recently, our group demonstrated that a higher
KYN/TRP ratio was significantly associated with greater depression scores in coronary artery
disease (CAD) patients, a relatable population to the stroke population41
. Taken all together,
these findings support the possibility of a biological contribution to PSD (alongside the current
psychosocial models) through IDO activation, whereby upregulation of IDO activity stimulates
the KYN pathway; thus depleting the amount of TRP available for 5-HT synthesis and
increasing the levels of KYN neurotoxins. A summary of KYN/TRP levels in psychiatric,
neurodegenerative, and inflammatory illnesses is displayed in Table 1.
_____________________________________
1.4.6 Rationale for this Study
Currently, no definite relationship has been established between pro-inflammatory
cytokine levels and PSD in humans. This study attempts to determine the relationship between
such and expects to find a positive causal relationship between plasma pro-inflammatory
cytokine concentrations and depression after stroke. In addition, this study also aims to examine
the counterintuitive reports of elevated anti-inflammatory cytokine levels192, 191
in post-stroke
patients. Instead, it is proposed that anti-inflammatory cytokines are involved in an innate
protective mechanism initiated by the body to counterbalance the depressiogenic effects induced
by pro-inflammatory cytokines. .
39
Finally, the study will examine IDO activity as measured by the KYN/TRP ratio. Since
TRP can only be obtained through diet, and since KYN371
and TRP compete with LNAAs for
entry through the BBB, peripheral measurements of both molecules should reflect the
concentrations circulating within the CSF. As summarized in the literature review, an elevated
KYN/TRP ratio will act as a marker for both possible neurotoxicity and decreased central TRP
availability. The additive affects of the neurotoxic KYN metabolites and reduced 5-HT
synthesis is predicted to play a significant role in the development of PSD. Indeed, increased
KYN/TRP ratio has recently been associated with depressive symptoms of other diseases (e.g.
post-partum depression, CAD); however, this will be the first study to examine its relationship
to PSD.
40
Table 1: Mean KYN/TRP Levels in Various Depression, Neurodegenerative Diseases, and Inflammatory Illnesses
Author Condition KYN/TRP Controls*
KYN/TRP Patients*
p-value Comments
Depression
Kohl39
Post-partum depression
29.4 ± 44.7 (n=86)
35.1 ± 47.5 (n=9)
0.067
KYN/TRP measured 4 days after birth; Edinburgh Postnatal Depression Scale (EPDS) score ≥ 12 positive for depression
Myint40 Major depression disorder
25.0 ± 11.0 (n=58) 17.0 ± 14.0 (n=189) 0.036 DSM-IV used for diagnosis of MDD; fasting blood levels drawn
Neurodegenerative Diseases
Forrest367
Huntington's
disease
33.8 ± 8.3 (n=11)
45.0 ± 7.9 (n=40)
<0.05
KYN/TRP significantly higher in only patients with the severest form of the disease (≥37 CAG triplet expansions on the gene)
Widner365
Alzheimer's
disease
34.1 ± 9.91 (n=20)
46.1 ± 19.8 (n=21)
0.018
AD patients with cognitive score lower than the median (<6; as measured by the Mini Mental State Examination (MMSE)) had significantly higher KYN/TRP (p=0.02) than those with MMSE≥6
Widner366
Parkinson's
disease
36.1 ± 8.2 (n=11)
58.5 ± 34.2 (n=7)
<0.05
Means displayed only compare controls to patients with the severest form of PD. Mean difference between controls and mild-moderate PD patients was non-significant
Inflammatory Illnesses
Bonaccorso38
Hepatitis C virus (Before and after IFN-α therapy)
39.0 ± 14.0
61.0 ± 31.0
<0.001
Patient group with IFN-α therapy, control group without IFN-α therapy. Treatment resulted in significantly higher depression scores at 4-6 months (p=0.03) as measured by the Hamilton Depression Scale (HAM-D)
*Reported as mean ± SD unless otherwise indicated
41
Author Condition KYN/TRP Controls*
KYN/TRP Patients*
p-value Comments
Inflammatory Illnesses
Lögters372
Major trauma (with or without
sepsis)
5.6 ± 0.4 (n=42)
21.9 ± 1.1 (n=17)
<0.01
KYN/TRPx100 reported here. Plasma levels measured 6 days after trauma; patient group is with sepsis, control group without sepsis
Huengsberg373
HIV
34.9 (32.4, 36.9)ɸ (n=72)
50.5 (44.9, 54.5)ɸ (n=82)
<0.01
Most patients were on treatment during the course of the study (53 patients took zidovudine (AZT); 23 took didanosine or zalcitabine)
Pertovaara374
Sjögren's syndrome
Females (n=139): 24.3 (21.0–28.9)
---------------- Males (n=7):
27.0 (23.6–32.1)
Females (n=170): 34.0 (25.1–44.3)
--------------- Males (n=170):
34.5 (29.3–38.7)
<0.0001
-------- 0.014
Patients were not age-matched; mean years control = 45 ± 11 years vs. mean years patients = 60 ± 12 years
Schroecksnadel363
Rheumatoid
arthritis
26.9 ± 8.10 (n=49)
37.9 ± 42.4 (n=22)
N/A
KYN/TRP tended to be higher in men than women, strongly associated with age (p<0.01), and significantly correlated with neopterin (p<0.05)
Swardfager41
CAD
(depressed vs. non-depressed)
38.5 ± 15.7 (n=24)
45.6 ± 20.0 (n=71)
0.055
All patients had CAD; control group represents those who were non-depressed, patient group represents those who were depressed
Wirleitner375
Coronary heart
disease
28.1 ± 5.25 (n=35)
36.3 ± 13.0 (n=35)
<0.01
KYN/TRP positively correlated with neopterin levels. Neopterin concentrations also positively correlated with older age
Zangerle376
HIV
30.7 ± 8.7 (n=40)
79.2 ± 60.3 (n=45)
<0.001
KYN/TRP significantly decreased (p<0.001) in patient group during anti-retroviral therapy (ART)
*Reported as mean ± SD unless otherwise indicated
ɸReported as median (95% confidence intervals) Reported as median (interquartile ranges)
42
Author Condition KYN/TRP Controls*
KYN/TRP Patients*
p-value Comments
Cancer
Schroecksnadel369 Gynecological cancer
35.1(19.2-59.0) (n=19)
41.4 (not reported) (n=20)
N/A
Patients with ovarian cancer had higher KYN/TRP trending on significance compared to controls
Suzuki360
Lung cancer
32.9 ± 9.10 (n=45)
47.1 ± 21.3 (n=123)
<0.0001
Higher KYN/TRP ratio was associated with advanced stages of the disease
Weinlich377
Malignant melanoma
26.9 ± 8.10 (n=49)
46.3 ± 20.7 (n=87)
<0.001
KYN/TRP and neopterin levels were positively correlated. Patients with KYN/TRP above the median (=41.2μmol/mmol) had a significantly decreased survival time compared to those above the median (p=0.0025)
*Reported as mean ± SD unless otherwise indicated Reported as median (interquartile ranges)
43
2. METHODS
2.1 Study Design
This was a cross-sectional observational study examining the role of several
neurochemical factors in the etiology of PSD. Patients meeting the World Health Organization
Multinational Monitoring of Trends and Determinants in Cardiovascular Disease (WHO-
MONICA) Project378
and National Institute of Neurological Disorders and Stroke (WHO-
NINCDS)379
criteria for stroke with CT or MRI evidence of acute ischemic infarcts were invited
to participate into the study. Based on the WHO-NINCDS guidelines, stroke was defined as ―a
sudden, nonconvulsive, focal neurologic deficit persisting for greater than 24 hours‖ and
excluded cases of transient ischemic attacks (TIAs). Five different health and rehabilitation
centres were utilized for recruitment: (1) Sunnybrook Health Sciences Centre, (2) Baycrest
Rehabilitation Centre, (3) St. John’s Rehabilitation Centre, (4) York Central Hospital and (5)
Toronto Rehabilitation Centre. Only patients who had an ischemic stroke within 12 weeks of
the initial assessment who spoke and understood English were considered for enrolment into the
study. If the patient fully agreed to the voluntary terms outlined for the study, a written
informed consent was completed (Appendix A). The individual then remained an active
participant until all consent terms were met unless the patient chose to terminate participation
prematurely.
At baseline interview, patients were considered depressed if they met the Diagnostic and
Statistical Manual of Mental Disorders, Fourth Edition (DSM-IV) criteria for depression, either
major or minor. Once screening tools and standardized mood questionnaires were completed, all
participants had blood samples drawn for cytokine, KYN, TRP, and LNAA analyses. In
addition, general cognitive function and stroke severity was examined in all consenting
participants.
44
2.2 Subjects
2.2.1 Inclusion and Exclusion Criteria
In all of the five listed recruitment sites, the study was approved by their respective
Research Ethics Boards (REBs) as shown by the most recent approval letters in Appendixes B
through F. The eligibility criteria for the study were carefully considered before approaching
patients and are described below:
Inclusion Criteria:
Age ≥18 years
Speaks and understands English
Clinical diagnosis of stroke according to WHO-MONICA378
and NINCDS379
criteria
Evidence of acute infarction on either CT or MRI
Maximum time since stroke onset <12 weeks
Written, informed consent
Exclusion Criteria:
Subarachnoid or intracranial hemorrhage
Severe aphasia or dysarthria that would interfere with the assessor’s ability to understand
patient response
Imminently suicidal or, in the opinion of the affiliated clinician, has inadequate family
monitoring for suicidality
Significant acute medical illness, including:
• Infection
Drug overdose
Alcohol abuse
Untreated hypothyroidism
Uncontrolled diabetes
Uncontrolled anemia
Severely disturbed liver,
kidney, or lung function
Significant acute neurologic illness, including:
Decreased Level of
Consciousness (LOC)
Active brain tumor
Parkinson’s disease
Huntington’s disease
Multiple sclerosis
Binswanger’s disease
Hydrocephalus
Subdural hematoma
Progressive supranuclear
paralysis
Severe aphasia
Presence of a premorbid Axis I psychiatric diagnosis, including:
Major depressive disorder
Schizophrenia
Bipolar disorder
Dementia
Concomitant use of psychotropics except for short acting benzodiazepines
47
2.2.2 Demographics and Medical History
Patient demographics including age, body mass index (BMI), level of education,
employment status, living status, marital status, history of depression, and number of vascular
risk factors (including hypertension, cigarette smoking, obesity, hyperlipidemia, and diabetes)
were either collected through patient interviews or medical histories stored on hospital
electronic databases. Current or past medical illnesses were determined through a review of
patient history, physical examinations, and/or routine laboratory test results by a licensed
practitioner as indicated in the patient charts. Finally, the time since stroke onset and
concomitant medications were recorded based on information gathered by the attending
physician upon admission.
_____________________________________
2.3 Clinical Assessments
2.3.1 Depression Scales
Centre of Epidemiological Studies Depression Scale (CES-D)
The Centre for Epidemiological Studies Depression Scale (CES-D)380
is a 20-item self-
rated questionnaire that was originally designed for use in community surveys as a means of
determining one’s depression quotient. Today, it is clinically employed as a screening tool for
depression and examines various aspects of the respondent’s perceived mood, anxiety and
physical functioning within the past seven days. However, because certain somatic symptoms
listed on the CES-D were originally intended to elicit symptoms of depression in otherwise
healthy individuals, the appropriateness of the scale requires validation among the stroke
population. Indeed, the scale has previously been validated in post-stroke patients using a
structured psychiatric interview as an established criterion. Here, a cut-off score of 16 was
found to be highly predictive of clinical depression, with a specificity of 90%, a sensitivity of
48
86%, and a positive predictive value of 80%381
. Thus, a score of 16 or greater was used as an
indicator of depressive symptoms in this study but may not have necessarily reflected definite
clinical depression. The CES-D has also been shown to exhibit high inter-observer reliability
and concurrent construct validity with several depression measures in the geriatric stroke
population (e.g. Geriatric Depression Scale (GSD), Zung Scale), as well as high discriminant
validity with measures of other factors including social functioning, cognition, and
disability382, 383
. As such, it has consistently been used as a screening instrument for
depression among the post-stroke population, including the NINCDS Stroke Data Bank384
and
other various PSD studies22, 385-387
.
Structured Clinical Interview for the DSM-IV: Depression Module (SCID)
In cases where patients scored 16 or greater on the CES-D, the attending psychiatrist
was called to further assess their depressive symptoms to ensure that they were not evoked by
the physical impairments suffered from stroke. The psychiatrist then confirmed a clinical
diagnosis of depression according to the DSM-IV criteria for a Major Depressive Episode
(MDE)388
, using the depression module of the Structured Clinical Interview for the DSM-IV
(SCID). The use of the SCID has previously been validated among the PSD population98, 99
.
Patients were considered to be suffering from major depression if at least five of the following
nine modules were met in the past two weeks: depressed mood, anhedonia, changes in weight
or appetite, insomnia or hypersomnia, psychomotor agitation or retardation, fatigue or loss of
energy, feelings of worthlessness or guilt, difficulties in concentration and decision-making, or
recurrent thoughts of death; with at least depressed mood or anhedonia present among their
symptoms. Patients were considered to be suffering from minor depression if they met three of
the nine modules, with at least depressed mood or anhedonia present among their symptoms.
Those who scored 16 or higher on the CES-D but failed to meet a formal diagnosis of
49
depression were placed in a "depressive symptoms only‖ group. However, the clinically
depressed group and the depressive symptoms only group were eventually combined for
analytical purposes due to the limitations of a small sample size. Thus, the patient population
used for analyses were not necessarily diagnosed with clinical depression. The CES-D has
consistently been used as a screening instrument for depression among the post-stroke
population, including the NINCDS Stroke Data Bank384
and other various PSD studies22, 385-
387. Even a briefer version of the CES-D that only included half of the original questions was
found to be a positive predictor of depression when compared to the DSM-IV criteria, with a
sensitivity of 97%, a specificity of 84%, and a positive predictive value of 85%
389.
2.3.2 Cognition Scales
Mini Mental State Examination (MMSE)
In addition to depression measurements, all patients were evaluated for general cognitive
function post-stroke. The Mini-Mental State Examination (MMSE)390
is the most is a popular
screening tool for this purpose and was administered to all recruited participants. It consists of
a brief, 30-point questionnaire for cognitive ability and evaluates various mental functions
including orientation, registration, short-term memory, attention, calculation, and visuo-spatial
skills. A patient who scored less than 24 was considered to be cognitively impaired and a
patient who scored 24 or more was considered non-cognitively impaired. Previously, the
MMSE has detected differences in concentration and memory function between depressed and
non-depressed stroke patients391
, as well as worse global cognitive function in PSD patients
with significant executive dysfunction392
. The MMSE has also been used to test the efficacy
of different antidepressants on reducing cognitive impairment in the PSD population128-130
.
50
2.3.3 Stroke Severity and Functional Disability
National Institutes of Health Stroke Scale (NIHSS)
Since the study was interested in examining the biological correlates related to PSD, it
was important to evaluate stroke severity and functional disability as possible confounding
factors to the results. To fulfill this task, The National Institute of Health Stroke Scale
(NIHSS)393
was administered to all stroke patients by the treating medical team or by a NIHSS
certified research associate. The NIHSS is a systematic assessment tool designed to
quantitatively evaluate the neurological deficits and degree of recovery of stroke patients. The
scale examines the following outcomes: level of consciousness, extraocular movements, visual
field defects, facial muscle function, extremity strength, sensory function, coordination
(ataxia), language (aphasia), speech (dysarthria), and hemi-inattention (neglect)394
. In this
study, the level of stroke severity was scored as follows: 0 = no stroke; 1-4 = minor stroke; 5-
15 = moderate stroke; 15-20 = moderate/severe stroke; 21-42 = severe stroke.
Previously, The NIHSS has demonstrated high intra- and inter-observer reliability to
non-neurologists and showed that study coordinators can be rapidly and reliably trained via
certification videos/DVDs to administer the NIHSS395, 396
. Furthermore, these certification
videos are reliable across multiple venues396
. NIHSS scores are also robust predictors of
stroke outcome and their values at baseline correlate strongly with the Trail of Org 10172 in
Acute Stroke Treatment (TOAST) stroke subtype diagnosis397
. In general, lacunar strokes are
related to lower NIHSS scores and thus, forecast better patient outcome. NIHSS scores are
also well correlated with infarct volumes, indicating strong concurrent validity of the scale398
.
_____________________________________
51
2.3.4 Blood Draw
Blood was drawn from inpatients by IV technicians at the time of their routine clinical
blood draws. Although blood orders were usually carried out in the morning (approximately
9:00 a.m.), there was no guarantee of a standardized collection time or that the desired blood
fasting levels were obtained. Serum levels of various pro- and anti-inflammatory cytokines
(IL-1β, IL-6, IL-10, IL-18, TNF-a, IFN-g) and kynurenine were analyzed, as well as plasma
levels of tryptophan and LNAA. Blood samples used for cytokine and kynurenine analysis
were collected in SST gel vacutainer tubes, while TRP and LNAA were collected in EDTA
vacutainer tubes.
Immediately after collection, blood samples were centrifuged at 1000g for 10 minutes.
Plasma or serum samples were transferred into labeled cryovials after centrifugation and
stored at -70°C until assayed. Because only the free fraction of plasma TRP reflects its
concentration in the brain, TRP samples required an additional ultrafiltration process to
separate its free form from its protein-bound form. Here, 1.0 cc of plasma was transferred into
an ultrafiltration device (Centrifree 4104®, Millipore) and centrifuged at 1000g for an
additional 15 minutes. Mechanistically, the device retained all protein-bound TRP while
allowing for the smaller free TRP to pass through the 30,000 kDa membrane into ultrafiltrate
reservoir cups. The cups containing the free TRP were then capped and stored with the other
samples at –70˚C until ready for batched analysis.
_____________________________________
2.4 Laboratory Analyses
2.4.1 Cytokines Assay
This study focuses on peripheral cytokine measurements. Several cytokines,
including IL-1α, IL-1β, IL-6, and TNF-α, are capable of crossing the blood brain barrier399
.
52
Although it cannot be positively confirmed that plasma cytokine levels are a direct reflection
of CSF levels, peripheral and central cytokine concentrations have been correlated in the
past400
. Finally, reproducibility of several past studies have indicated plasma cytokines to be
reliable markers of depression175, 401
and stroke outcome171, 191, 192, 400, 402
.
Cytokine measurements of IL-6, IFN-γ, and I-10 were performed by Dr. Angela
Panoskaltsis-Mortari who heads the Cytokine Reference Laboratory at the University of
Minnesota. The technique employed for all three cytokines was multiplex immunobead-based
assay using the Human Fluorokine Multianalyte Profiling (MAP) Base Kit A (R&D Systems,
Minneapolis, MN). This bead-based assay only requires a small sample size, allows the user
to define specific molecules of interest, and is known to be highly efficient and flexible for the
simultaneous detection of process-related molecules via Luminex xMAP® technology. Each
sample has been evaluated for cytokine sensitivity, precision, recovery, sample linearity, and
specificity by the company panel. A brief mechanistic summary of the technique is provided
as follows from the company website:
"Detection is achieved through a bead-based antibody-antigen sandwich
method. Briefly, samples are incubated with color-coded beads that are pre-
coated with analyte-specific capture antibodies for the molecule of interest.
Expression levels are determined following incubation with a biotinylated
detection antibody and streptavidin-conjugated phycoerythrin (PE). Using a
Luminex® analyzer, independent lasers determine the color of each bead
and the magnitude of the PE-derived signal, which is directly proportional to
the levels of bound analyte."
The procedure is similar to that described by Djoba Siawaya et al403
, who evaluated
and compared several commercial bead-based luminex cytokine assays. The group reported
that R&D Systems Fluorokine-MAP assays were one of the most accurate for the
measurement of cytokine concentrations in whole blood culture supernatant and achieved
good recovery ranges and reproducibility for most cytokines. According to the data provided
53
by R&D Systems, assay sensitivities were 1.11 pg/mL for IL-6, 0.3 pg/mL for IL-10, and 1.27
pg/mL for IFN-γ. A more detail description of assay sensitivities as described as the minimum
detectable dose is shown in Table 2 below:
Table 2: Cytokine Assay Sensitivities
Cytokine Number of Assays MDD range MDD mean
IL-6 43 0.10 - 1.11 pg/mL 0.36 pg/mL
IL-10 43 0.07 - 0.30 pg/mL 0.13pg/mL
IFN-γ 42 0.09 - 1.27 pg/mL 0.31 pg/mL
2.4.2 KYN Assay
The KYN assay was performed by the Pharmacy Quality Control and Research Group at
Sunnybrook Health Sciences Centre. The technology utilized was reverse phase high performance
liquid chromatography (HPLC) at UV detection of 258nm. The exact steps taken for the KYN assay
were provided by Scott Walker in a personal communication. Briefly, the method for KYN
involves protein precipitation with an equal volume of 3% perchloric acid. The sample was
centrifuged for 10 minutes and 0.1 mL of the supernate was injected directly into the high
liquid chromatographic system using an autoinjector (715 Ultra WISP, Waters Canada). The
mobile phase consists of 9% acetonitrile in 0.05 M potassium phosphate mono basic, which
was pumped through a reverse phase 5 µm ODS column , 250 mm x 4.6 mm (Symmetry;
Waters Corp) at 1.0 mL/min using a chromatographic pump (P4000; ThermoSeparation
Systems, San Jose CA). KYN was detected using an ultraviolet detector (UV 6000;
ThermoSeparation Systems, San Jose CA) at a wavelength of 258nm. Chromatograms were
recorded on a computer using the ChromQuest software (ThermoQuest, San Jose CA).
The standard curve was constructed by creating 9 standards from a common serum
stock sample. To approximately 10 mL of serum, additional KYN was added to the KYN
present in the sample to create standards with 0, 0.125, 0.250, 0.3125, 0.50, 0.75, 1.0, 1.5 and
2 mg/L of additional KYN added. Analysis of these standards in duplicate resulted in linear
54
relationship between the KYN peak area and concentration with a slope and intercept (due to
the endogenous kynurenine in the common serum stock). Unknown concentrations of KYN in
plasma were determined by dividing the KYN peak area in the unknown sample by the slope.
Replicate error, as measured by the coefficient of variation (CV(%) = 100 x standard
deviation/mean), based on duplicate analysis of standards between 0 mg/L of kynurenine
added to 2 mg/L added ranged from 1.54% to 0.5% with deviations from the known or
expected concentrations averaging less than 7%.
2.4.3 TRP and LNAA Assay
The TRP and LNAA assays were performed by Dr. Simon Young in the Department of
Psychiatry at McGill University who used the same procedure as described by Anderson et
al404
. Details of the TRP and LNAA assays were provided by Simon Young via personal
communication:
―Plasma was deproteinized by adding one part of 1 M perchloric acid to
three parts of plasma, mixing and centrifuging. Tryptophan in plasma
was measured by high-performance liquid chromatography (HPLC) on
a Waters Bondapack C18 reverse phase column (Phenomenex,
Torrance, California) with fluorometric detection (Anderson and Purdy,
1979). The other large neutral amino acids (LNAA) (tyrosine,
phenylalanine, leucine, isoleucine, valine) were measured on a
Beckman System Gold amino acid analyzer using precolumn
derivatization with o-phthalaldehyde and gradient reverse phase HPLC
with fluorometric detection. Aminoadipic acid was used as an internal
standard.‖
_____________________________________
2.5 CT Scan Analysis
The majority of patients admitted to Sunnybrook for acute stroke had CT scans
performed within the first 24 hours of admission. Lesion characteristics were determined from
CT scans obtained without a contrast agent on a General Electric LightSpeed VCT series
scanner (General Electric Healthcare, Waukesha, WI). Ischemic lesions were manually traced
55
on these images using Medical Image Processing, Analysis, and Visualization (MIPAV;
National Institutes of Health, Bethesda, MD). Lesion area was measured with the use of a
digitizing tablet (Sigma Scan, version 3.0, Jandel Scientific) from tracings of the infarct on
each slice of the CT scan on which the lesion appeared. The area of the lesion on each slice
was multiplied by the slice thickness (1 mm3) and summed to obtain the total lesion volume.
The criteria for anterior lesion was that its anterior most border must extend in a rostral
manner past the 40% anterior-posterior (A-P) distance. The criterion for posterior lesion was
that its posterior border must extend in a caudal manner past the 60% A-P distance. Lesions
that fell between the 40% and 60% A-P distance were classified as intermediate. Lesions that
eclipsed both the 40% and 60% A-P distances was classified as extending. Infarct side (left
vs. right) was determined by examining which side the lesion laid to the longitudinal fissure
on the CT scans.
_____________________________________
2.6 Statistical Analyses
The Statistical Package for the Social Sciences (SPSS), version 17.0 was used for all
statistical analyses.
2.6.1 Preliminary Data Transformations
NIHSS scores are supposed to be collected upon patient admission and entered into the
chart. However, 23 scores were missing from the charts. The NIHSS score was imputed at the
group mean for both the control and depression group (since the stroke scale was not
performed on all patients) in order to keep a consistent sample size for analyses. NIHSS
scores were collected based on forms that were filled out by the attending medical staff upon
patient admission. Thus, about half of all scores were missing as some of the NIHSS forms
were not contained within the patient charts. In total, 23 NIHSS scores were imputed—18
56
from the control group and 5 from the depressed group. In the case of the MMSE, a percent
score was calculated for all patients who could not complete the entire examination due to
physical impairments from stroke (e.g. motor injuries would void the individual form drawing
and writing activities), where the correct number of responses was divided by the largest
possible score that can be obtained during assessment. The percent was then multiplied by 30
(the standard MMSE total score) to maintain an invariable range of scores and the method has
been validated among the elderly population405, 406
. Additionally clinical measurements of
cytokine assays were sometimes returned with undetectable levels or below the lowest level of
detectability of the assay. When this occurred, the lowest level of detectability for each
cytokine (1.11 pg/mL for IL-6, 0.30 pg/mL for IL-10, and 1.27 pg/mL for IFN-), was used as
the imputed value.
Although this method would generate cytokine values that deviate from the true mean,
the main goal of this study was not to capture low concentrations of pro-inflammatory
cytokines but their elevated levels. Thus, the approach should not affect our ultimate intent of
finding clinically significant elevated cytokine concentrations. However, data pertaining to
anti-inflammatory cytokines may be affected since our hypothesis predicts the presence of
decreased concentrations. This limitation will be explored further in the discussion.
2.6.2 Planned Analyses
Clinically depressed patients and those who only exhibited depressive symptoms
(CES-D≥16) were combined into one group and collectively labeled the "depressed group".
As mentioned previously, the number of patients in the study with major versus minor
depression made subanalysis by depression subtype unfeasible. Thus, throughout this paper,
the term "depressed group" will be used to represent the combined depressed and depressive
symptoms group. Those who did not exhibit any clinically significant signs of depressive
57
symptoms (CESD≤15) were labeled the "non-depressed group". Complete demographic data
were collected during recruitment and compared between the two groups using independent
samples t-tests for continuous data (e.g. age, BMI) and chi-square or Fisher's exact tests for
categorical data (e.g. marital status, employment status, living situation). Variables that were
significantly different between groups were included as covariates in subsequent analyses.
2.6.2.1 Primary Analysis
Hypothesis 1: Increased idoleamine 2,3-dioxygenase (IDO) enzyme activity will be found in
stroke patients experiencing depressive symptoms compared to non-depressed stroke patients
as evidenced by an increased kynurenine/tryptophan (KYN/TRP) ratio.
Demographic and clinical characteristics were compared between depressed and non-
depressed groups using independent samples t-test for continuous variables and Pearson's chi-
square analysis for categorical variables. ANCOVA was used to compare differences in mean
KYN/TRP ratio between groups with any between-group demographic or clinical differences
entered as covariates.. Depression was also analyzed as a continuous variable by looking at the
relationship between CES-D scores and KYN/TRP using a partial correlation.
2.6.2.2 Secondary Analyses
Hypothesis 2: Increased levels of pro-inflammatory cytokines (IL-6, IFN-) and decreased
levels of anti-inflammatory cytokines (IL-10) will be found in stroke patients experiencing
depressive symptoms compared to non-depressed stroke patients.
Skewness and kurtosis were examined for each cytokine variable. If the data presented
itself as unskewed, ANCOVA was used to compare differences in mean cytokine levels
between depressed and non-depressed patients, with the appropriate demographic variables
acting as covariates as determined during preliminary analysis. If the data presented itself as
being skewed, a non-parametric Mann-Whitney test was employed instead to determine the
58
mean cytokine difference between patient groups. Additionally, Spearman's rank rho were
performed between all cytokines and the KYN/TRP in order to determine the relationship
between the two variables.
2.7 Sample Size Calculations
This is the first study to evaluate the relationship between KYN/TRP ratio and its
predictive value of depression post-stroke. Thus, estimates of effect size were based on
previously published data examining the relationship between KYN/TRP ratio and depression
in CAD patients41
. Since the primary analysis of this study was based on ANCOVA statistics,
sample size calculations for the multivariate data were not possible as no supportive data was
available for this method407
. Thus, all effect estimates were based on the procedure for
independent samples t-tests. Based on the CAD study, mean KYN/TRP ratios and the
weighted standard deviation were used in the equation described as follows. Effect size (d)
were calculated according to the equation d = (mA – mB) / σ; where mA = the population mean
for group A (MDD patients), mB = the population mean for group B (non-depressed controls),
and σ = the population standard deviation. Required sample size was calculated according to
the equation n = 2 [ (zα + zβ) σ / Δ ]2 or n = 2 [ (zα + zβ) / d ]
2 where Δ = (mA – mB), the
difference between the two population means, zα = the z-value that corresponds to the
accepted α level (probability of type I error), and zβ = the z-value that corresponds to the
accepted β level (probability of type II error). For α = 0.05, zα = 1.96 and for β = 0.20, zβ =
0.84. It was determined that for α = 0.05, 88 patients per group would be necessary to obtain
an observed power of 0.80.
_____________________________________
59
2.8 Control of Confounders
Both depression and stroke have been individually and collectively linked to several
clinical and cardiovascular risk factors in the past research studies. For this reason, potential
confounders were compared between depressed and non-depressed patients. Based on the
statistical analyses, variables found to be significantly different between groups were
considered as covariates in the primary analyses. Only hypertension and levels of LNAA
were found statistically significant among all measured demographic and clinical factors. As
previously mentioned in 1.4.1.5 hypertension has previously been found in subjects with
depressive symptoms110
and thus may be pertinent to the development of PSD.
3. RESULTS
Patient recruitment began at the inpatient stroke unit at Sunnybrook Health Sciences
Centre in July 2007. A list of possible study participants was provided weekdays by a nurse
responsible for keeping track of admitted stroke patients. This list of potential research
subjects is generated as part of the Heart and Stroke Foundation Centre for Stroke Recovery
research initiative. Potential recruits were subsequently approached by the study coordinator
to ask if they were willing to participate in a research study. The coordinator described the
premise of the study, what was required of the patient if he or she chose to participate, and the
risks and benefits of those requirements. However, because the research is complex in its
variability, the ability for each individual to understand the research also varies. Thus, the
capacity for consent was verified by independent physicians for all patients as part of standard
care. As a further precaution, the assessor had the patients repeat the purpose and details of
the study to ensure that they were fully aware of their responsibilities with participation.
Finally, the study requires consenting to questionnaires and a blood sample, which are not
60
complicated tasks to understand in themselves. A flowchart of patient recruitment progress
can be viewed in Figure 2 along with a table of ineligibility in Table 3.
Figure 2. Patient Recruitment
Ischemic Stroke Patients n=489
Minimum Length of Stay ≥5 days n=346
Screened n=303
Non-Depressed n=38
Depressive Symptoms n=16
Eligible n=125
Not Eligible n=178
Recruited n=61
Completed n=54
No Consent n=64
61
Table 3: Participant Ineligibility
Reason for Ineligibility Number of Patients
History of Depression 34
Non-English Speaking 29
Severe Aphasia/Dysarthria 19
Decreased LOC 15
Neurological Disorder
Dementia 18
Parkinson’s Disease 5
Other 9
Medical Illness
Infection 13
Rheumatoid Arthritis 3
Other 6
Other 27
Total 178
At the time of this paper, 61 patients have agreed to participate in the study with
recruitment ongoing. All individuals who were selected as participants met the eligibility
criteria, signed a written informed consent and provided a blood sample for biomarker
analyses. However, due to loss of follow-up, only 54 (38 non-depressed, 16 depressive
symptoms) were used for analyses. Based on these numbers, the study group consists of 30%
of stroke patients experiencing significant depressive symptoms, which is an agreement with
previously published results regarding the prevalence of PSD10
.
_____________________________________
3.1 Demographic and Clinical Characteristics
All pertinent demographic and clinical characteristics are reported in Table 4 via
independent t-test and chi-square analyses. It is uncertain how long a patient was depressed
because the diagnosis of depression was made by our team. However, patients with a prior
history of depression or who were on anti-depressants at the time of approach were excluded
62
from the study. Therefore, it is unlikely that the depression evaluated by our team was a result
of a previous history of depression rather than the events prompted by stroke.
Table 4: Participant Demographics and Assessment Scores by Depression Group
Non-depressed (n=38)
Depressive Symptoms (n=16)
p-value
Demographics
Sex 52.6% 50.0% 0.860
Age 71.3 ± 17.7 69.4 ± 14.3 0.674
BMI (kg/m2) 28.0 ± 4.8 26.1 ± 5.4 0.228
Time between date of stroke and assessment (days)
30.2 ± 42.0 25.6 ± 36.8 0.713
Marital status (% married) 39.5% 68.8% 0.233
Living situation (% living with others)
68.4% 75.0% 0.751
Employment (% retired) 76.3% 75.0% 0.286
Education (% with Bachelor’s degree or greater)
36.8% 18.8% 0.514
Assessment Scores
CES-D score 6.2 ± 5.0 26.8 ± 10.8 <0.0001
MMSE imputed score 26.6 ± 4.3 25.55 ± 4.7 0.090
NIHSS score 7.6 ± 5.6 11.4 ± 5.4 0.097
Blood Markers
LNAA (µg/mL) 578.8 ± 147.0 538.6 ±141.2 0.022
KYN (µmol/L) 2.8 ± 1.6 3.3 ± 1.6 0.582
TRP (µg/mL) 8.8 ± 2.5 9.3 ± 3.7 0.120 For continuous variables, data is represented as mean ± SD
No significant differences were found between patient groups except for hypertension
(p=0.043). Although PSD is often found comorbid with cognitive impairments, the current
patient population did not display such a relationship as no significant difference was found
between mean MMSE scores (non-depressed group=26.6±4.3, depressed group=25.6±4.7,
F2,49=2.53, p=0.090). Plasma markers were examined using ANCOVA analysis with
hypertension acting as the covariate. Mean LNAA concentrations were found to be
significantly lower in the depressed patient group (F2,51=4.10, p=0.022), while other plasma
markers including TRP and KYN were found to be non-significant.
63
Lesion characteristics of stroke patients with and without depression can be viewed in
Table 5. The mean stroke lesion volume among all stroke patients was 21.89 ± 50.8 cm3 but
was not significantly different among non-depressed and depressed stroke patients (non-
depressed=23.6 ± 58.3, depressed=18.3 ± 31.8, p=0.763). The majority of stroke patients
(38.5%) had lesions located in the intermediate region (between anterior and posterior
lesions). The rest of the patient population had lesions in the following locations: 35.9% of in
the anterior circulation, 23.1% in the posterior circulation, and 2.6% extended from the
anterior circulation to the posterior circulation. There was no significant difference in lesion
locations between groups (2=3.74, p=0.291). Among all patients, 54.9% had right-sided
lesions, 43.1% had left-sided lesions, and 2.0% had lesions spanning both the left and right
sides. Finally, there was no significant difference in infarct side between depressed and non-
depressed patients (2=0.49, p=0.781).
Table 5: Stroke Lesion Characteristics by Depression Group
Non-depressed (n=27)
Depressive Symptoms (n=13)
p-value
Volume (cm3) 23.6 ± 58.3 18.3 ± 31.8 0.763
Location • %Anterior • %Posterior • %Intermediate • %Extending
34.6% 30.8% 30.8% 3.8%
38.5% 7.7% 53.8%
0%
0.291
Laterality (% Right) 59.3% 61.5% 0.781 For continuous variables, data is represented as mean ± SD
Almost all patients had at least one cardiovascular risk factor based on the entire study
population, including hypertension (71.4%), hypercholesterolemia (37.0%), diabetes (18.5%)
or cigarette smoking (18.5%). All recorded cardiovascular risk factors were found to be
higher in the depressed patient population as displayed in Table 5; however, only the
proportion of hypertensive patients in the depressed groups was significantly greater than the
non-depressed group (93.8% vs. 65.8%, χ2=4.48, p=0.04).
64
Based on different medical histories, all patients were treated with a variety of
medications including anti-hypertensives (57.4%), aspirin (66.7%), beta-blockers (37.0%),
calcium channel blockers (29.6%) and diuretics (16.7%). The proportion of patients taking a
certain medications did not differ between patient groups. Although the regular use of
psychotropics was counted as an exclusion criterion during recruitment, short acting
benzodiazepines (e.g. lorazepam) were permitted in this study to sedate or to aid patients in
their sleep. Medications consumed by both patient groups by proportion are displayed in
Table 6 via chi-square analysis.
Table 6: Cerebrovascular Risk Factors and Concomitant Medication Use by Depression Group
Non-depressed (n=38)
Depressive symptoms (n=16)
p-value
Cerebrovascular Risk Factors
Hypertension 65.8% 93.8% 0.043
Hypercholesterolemia 34.2% 43.8 0.507
Obesity (BMI ≥ 30) 31.6% 25.0% 0.629
Diabetes 15.8% 25.0% 0.426
Cigarette smoking 18.4% 18.8% 0.977
Total number of CVRFs 1.7 ± 1.2 2.1 ± 1.1 0.198
Concomitant Medications
Aspirin 68.4% 62.5% 0.673
-blockers 36.8% 37.5% 0.964
Ca+2 channel blockers 23.7% 43.8% 0.140
Diuretics 15.8% 18.8% 0.790
Insulin 15.8% 25.0% 0.426
Benzodiazepines 13.2% 6.3% 0.461 For continuous variables, data is represented as mean ± SD
_____________________________________
3.2 Assay Results
A total of 54 plasma samples (38 non-depressed, 16 depressive symptoms) were
collected and sent for analysis. Plasma markers of interest were KYN, TRP, LNAA and the
cytokines IL-6, IL-10, and IFN-. However, several cytokine samples were below the lower
limit of the assay’s detectability. As previously mentioned, these limits were 1.11 pg/mL for
65
IL-6, 0.30 pg/mL for IL-10, and 1.27 pg/mL for IFN-. Thus, imputed scores were also used
for cytokine statistical analyses, where the lowest level of detectability was used as the
imputed values.
3.3 Biological Correlates of Post-stroke Depression
3.3.1 Hypothesis 1: KYN/TRP ratio as a marker of depression
As displayed in bar graph form in Figure 3, ANCOVA analysis revealed a significant
difference in mean KYN/TRP ratio between depressed and non-depressed patient groups with
hypertension and LNAA concentrations as covariates (non-depressed group=69.32±36.90,
depressed group=78.33±42.03, F3,50=4.61, p=0.006) and an observed power of 86.4%.
Figure 3. Mean KYN/TRP ratios in stroke patients with and without depressive symptoms
* F3,50=4.61, p=0.006; non-depressed=69.3±36.9; depressive symptoms = 78.3±42.0 Values reported as mean ± SD.
A box-plot displaying further differences in the range, median, and interquartile ranges
of the KYN/TRP ratios can be viewed in Figure 4. However, partial correlation analysis
revealed a non-significant relationship between CES-D scores and KYN/TRP while
66
controlling for hypertension and disparities in LNAA concentrations (r50=-0.06, p=0.67). A
scatter plot outlining the relationship between CES-D scores and KYN/TRP ratio is displayed
in Figure 5.
Figure 4. Box-plot of unadjusted KYN/TRP ratios in stroke patients with and without depression
Figure 5. Correlation between CES-D total scores and KYN/TRP ratio
The same ANCOVA analysis was conducted after separating the combined depression
group into ―depressed‖ and ―depressive symptoms only‖ groups such that a total of three
67
groups existed. The results remained significant between groups (F4,49=4.027, p=0.007),
where those in the depressive symptoms category had a much higher KYN/TRP that the other
two groups. These results suggest that KYN/TRP may act as a red-flag for those most prone to
depressive symptoms post-stroke and possibly developing clinical depression thereafter. No
comment can be made about the depressed group due to the small sample size.
Other exploratory analysis found a significant correlation between KYN/TRP levels
and LNAA concentrations using bivariate correlation analysis (r2=0.21, p<0.001). A scatter-
plot of the relationship can be viewed in Figure 6.
Figure 6. Correlation between LNAA concentrations and KYN/TRP ratio
The presence of hypertension between depressed and non-depressed patients was
explored further since chi-square analysis found a significantly greater number of hypertensive
patients in the depressed than the non-depressed population via chi-square analysis (93.8% vs.
65.8%, χ2=4.48, p=0.03). Unfortunately, the production of a correlative relationship between
the two variables was not possible since systolic pressure over diastolic pressure values were
not collected during recruitment. To observe the reverse relationship where groups were
68
separated by the presence of hypertension rather than depression, ANOVA analysis revealed a
significant difference in mean CES-D scores between hypertensive and non-hypertensive
patients (non-hypertensive=7.0 ± 5.7, hypertensive=14.2 ± 12.9, F1,52=3.9, p=0.05). A graph of
this relationship can be viewed below in Figure 7.
Figure 7. Mean CES-D Scores by Hypertension Group
Further exploratory analysis was conducted to determine the relationship between
hypertension and the KYN/TRP, the KYN/TRP values were first separated into two groups,
―low‖ vs. ―high‖ (based on the median), proceeded by chi-square analysis with hypertension
acting as the other variable. However, results were non-significant (percent non-hypertensive
above the median KYN/TRP ratio=50% vs. percent hypertensive above the median KYN/TRP
ratio=73%, χ2=2.36, p=0.12). When ANOVA was conducted using the same variables, the
results were no different in significance between hypertension and mean KYN/TRP ratio
(mean KYN/TRP non-hypertensive=59.5±41.7, hypertensive=76.3±36.6, F1,52=2.3, p=0.16).
Although the relationship between hypertension and KYN/TRP was not significant,
hypertension acted as a significant co-variate in our analysis of KYN/TRP and depression
69
group. Thus, the effect of anti-hypertensive medication on the results of this primary
hypothesis was also examined. Hypertension was diagnosed based on reviewing the past
medical history in the patient chart. Out of all 54 patients, 40 were hypertensive and 31
patients were on anti-hypertensive medication. The distribution of hypertensive/non-
hypertensive patients on anti-hypertensive medication is listed in Table 7 as follows:
Table 7: Distribution of Hypertensive Patients on Anti-Hypertensive Medication
Hypertensive Non-hypertensive
On anti-hypertensives 27 4
Not on anti-hypertensives 13 10
Those who did not have a history of hypertension but were currently placed on anti-
hypertensives as an inpatient may have acquired hypertensive characteristics because of
complications from stroke. Likewise, those who had a history of hypertension but were not
give anti-hypertensives as an inpatients may have been taken off the medication due to
medical complications as seen fit by the attending doctor. When both hypertension and the
use of anti-hypertensive were controlled for in analysis of the primary hypothesis, The
KYN/TRP ratio remained significantly higher in the depressed group (F4,49=4.0, p=0.007).
Thus, the presence or absence of anti-hypertensive medication did not significantly alter our
results.
3.3.2 Hypothesis 2: Inflammatory markers of depression
Because preliminary analysis found the cytokine data to be highly skewed, a Mann-
Whitney test was employed to compare the mean cytokine levels between depressed and non-
depressed stroke patients with hypertension acting as the covariate. All three measured
cytokines (IL-6, IL-10, IFN-) revealed non-significant results (p=0.32, p=0.19, p=0.21)
respectively. When the imputed values were used for analysis, results remained non-
significant (p=0.627, p=0.216, p=0.297) respectively. However, about half or more of the data
70
required imputation creating a skewed set of data that would be hard to detect significance
even with non-paramatric tests. Nevertheless, the values remained numerically in the
direction of expectation, where pro-inflammatory cytokine levels were greater in depressed
group and the anti-inflammatory cytokine levels were lower in the depressed group. A more
detailed summary of the results can be viewed in Table 8.
Table 8: Measured and Imputed Cytokine Plasma Levels by Depression Group
N Non-depressed Depressed p-value*
Unadjusted values
IL-6 (pg/mL) 30 7.9 ± 5.0 9.3 ± 3.7 0.329
IL-10 (pg/mL) 13 1.9 ± 1.5 1.0 ± 0.4 0.192
IFN- (pg/mL) 27 5.1 ± 3.7 5.3 ± 1.7 0.205
Imputed values
IL-6 (pg/mL) 54 4.9 ± 5.0 5.7 ± 5.0 0.627
IL-10 (pg/mL) 54 0.59 ± 0.86 0.56 ± 0.42 0.216
IFN- (pg/mL) 54 3.1 ± 3.2 3.5 ± 2.4 0.297
*Based on Mann-Whitney statistics
Data is represented as mean values ± SD
Due to the high number of samples lost as a result of undetectability, chi-square
analysis was conducted to compare if there was a significant difference in percentage of
cytokine detectability between groups depression group. This was not the case as displayed in
the table below. Thus, detectability of cytokines should not have affected our results and was
not treated as a covariate during any analyses.
Table 9: Cytokine Assay Detectability by Depression Group
Non-depressed Depressed 2 p-value*
IL-6 56.8% 56.3% 0.001 0.973
IL-10 18.4% 37.5% 2.242 0.134
IFN- 47.4% 56.3% 0.355 0.551
Spearman's rank rho analysis revealed a relationship trending toward significance
between measured unadjusted IL-6 concentrations and KYN/TRP ratio (rho=0.33, p=0.08) and
is exhibited in scatter-plot form in Figure 8. However no other cytokine resulted in a similar
relationship with the KYN/TRP ratio. In addition, non-significant correlations were found
71
between all cytokines and total CES-D score when both the raw and imputed data were used
for analysis.
Figure 8. Correlation between unadjusted IL-6 concentrations and KYN/TRP ratio
Finally, the magnitude of variability imposed by combining the depression groups was
observed. When the combined depression group was separated into ―depressed‖ and
―depressive symptoms only‖ such that a total of three groups existed rather than two, the
results did not significantly differ from the originally reported results after running the same
analysis. The time of blood collection with respect to stroke onset was also evaluated for its
variability. Only 10 of the total 54 patients had blood drawn within greater than 1 month post-
stroke, 2 of which were in the depressed group. However, our preliminary analysis showed
that time since stroke and enrollment into the study did not significantly differ between patient
groups, thus, the inclusion of patients >3 months post-stroke should not significantly affect our
results. Nevertheless, when the 10 patients were removed from the subject population and the
cytokine analysis were rerun, they did not significantly differ from our initially reported
results.
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4. DISCUSSION
This study is the first to support the role of IDO activation in the etiology of PSD via
the KYN/TRP ratio marker. Mean KYN/TRP ratio was significantly higher in depressed than
non-depressed stroke patients. However, the severity of depressive symptoms, as measured by
total CES-D score, did not significantly correlate with KYN/TRP ratio or plasma cytokine
levels. Additionally, cytokine levels did not significantly differ between patient groups, but a
correlation trending on significance was found between KYN/TRP and IL-6 plasma levels.
Finally, the percent of hypertensive patients was significantly higher in depressed stroke
patients, a finding supported by a higher mean CES-D score among hypertensive patients than
non-hypertensive patients. No significant difference in mean KYN/TRP was found between
hypertensive and non-hypertensive patients.
_____________________________________
4.1 The Role of IDO Activation in Post-stroke Depression
Several studies examining neurological illnesses associated with unstable levels of
inflammatory markers have reported elevations in KYN/TRP ratio, including in Alzheimer’s
disease365
, Parkinson’s disease366
, HIV infection408
, rheumatoid arthritis363
, coronary heart
disease41, 375
and healthy aging409, 410
. Additionally, an increase KYN/TRP ratio has also been
associated with states of depression including women with post-partum depression39, 370
and
depressed hepatitis C and cancer immunotherapy patients38, 337
. However, despite several
speculations of a causal relationship between activation of the KYN pathway and the
development of PSD by various research groups, no association has been made to date. This
study is the first to report a significant difference in mean KYN/TRP ratio between depressed
and non-depressed stroke patients with a high observed power of 86.4%. Although this result
73
was relatively surprising since the sample size calculation predicted that 88 patients per group
was required to fulfill an observed power of 80%, we were still able to demonstrate
significantly elevated KYN/TRP ratios among depressed patients.
The results presented in this study agree with our hypothesis that IDO is activated upon
inflammatory burden post-stroke. Because the IDO enzyme has an extremely short half-life,
direct measurement of its plasma concentrations was not feasible. Thus, the ratio of its
primary metabolite KYN to TRP was used as a marker of its activity instead. An increase in
IDO activity may reduce 5-HT production since the enzyme is in direct competition with TPH
for the metabolism of TRP into 5-HT. Consequently, central 5-HT levels are decreased below
the level for normal mood and behavioural regulations. For example, TRP levels have been
found to be decreased in depressed patients314
while the administration of TRP produces an
antidepressant effect340
. Thus, depletion of TRP as a result of increased IDO activity may
explain our finding that the depressed patient population had a significantly higher KYN/TRP
than non-depressed subjects.
Increases in KYN levels may also affect mood since it can be further metabolized into
several neurotoxic metabolites including three potent neurotoxins; the hydrogen peroxide
generators, 3-hydroxykynurenine and 3-hydroxyanthranilic acid223
and the NMDA agonist,
quinolinic acid (QUIN)335
. Because these intermediates are neuroactive, central elevations in
their concentrations may destroy essential neurons responsible for controlling mood and
behaviour within the limbic structures of the brain. For example, 3-hydroxykynurenine levels
have been found to be elevated in other neurological disorders such as Huntington’s disease
patients354, 355
and has been shown to induce neuronal cell death in cortical and striatal cell
cultures44
. Intracerebral injection of 3-hydroxyanthranilic acid can decrease choline
acetyltransferase activity225
thus, decreasing acetylcholine synthesis. Finally, several
74
neurodegenerative conditions have been linked to QUIN-induced apoptosis, including
Huntington’s disease357
and HIV-associated dementia358
. Furthermore, laboratory studies
have demonstrated the production of QUIN to be immune mediated (in this case, stroke) and
acts as a potent agonist of the neuronal NMDA subtype of glutamate receptors335
. It is thus no
surprise that a significantly elevated KYN/TRP ratio was detected in the depressed patient
population as a combination of both decreased serotonin synthesis and increase neurotoxic
KYN metabolites can result in the clinical manifestations of PSD. Also note that stroke
severity was not significantly different between groups, suggesting that the described
biological pathway may be partially responsible for PSD rather than the sole influence of
post-stroke physical impairments.
An effort was made to control the potential demographic confounders collected during
the recruitment period. The presence of both hypertension and low LNAA concentrations
significantly differed between patient groups and were thus used as covariates in the analyses.
Properties of hypertension including decreased lumen size and decreased vessel distensibility
have previously been found to be prominent in patients with depressive symptoms and those
who also suffer from reduced blood flow velocity were diagnosed with ad DSM-IV depressive
disorder110
. LNAA directly competes with TRP for transport into the brain and because the
direction of TRP metabolism affects synthesis of 5-HT and KYN, significantly different levels
of LNAA would be expected from depressed patients.
In this study, plasma levels of LNAA were significantly lower in depressed patients
while TRP levels (although not significant) were numerically higher. This counters past
studies that have reported lower TRP and TRP/LNAA ratio in MDD patients compared to
healthy controls305, 310-312
. Our contrasting results may include one of the following three
explanations, a combination of all, or other factors outside our control. First, it is common for
75
clinical studies to measure plasma TRP in the morning after overnight fast since TRP can only
be obtained exclusively through dietary sources. Although our study attempted to ensure all
blood samples were collected in the morning (approximately 8 a.m.) during the IV
technologist’s regular rounds, patients were known to have breakfast as early at 7 a.m. in some
cases, while blood draw times were delayed to approximately 11 a.m. in other cases. Thus,
variability in collection time and diet may have contributed to the unexpected increase in TRP
availability among stroke patients. Secondly, the amount of dietary tryptophan and LNAAs
(e.g. tyrosine) consumed may differ between patient groups depending on their willingness to
eat. Since one of the most defining symptoms of depression includes the loss of appetite,
variations in TRP and LNAA consumption between depressed and not depressed patients may
have confounded our results. Finally, peripheral levels of TRP, KYN, and LNAA may not
parallel with its central availability. It would be necessary to devise experiments looking at
both the central and peripheral availability of these molecules in order to validate if an
increased TRP/LNAA ratio or its opposite is representative of the observed depressive
symptoms in PSD patients.
Interestingly, a significant positive correlation could not be found between total CES-D
scores and the KYN/TRP. Therefore, it would be necessary to test this hypothesis on a greater
sample population of depressed patients as our study was limited to a small group of subjects.
This was mostly due to a decrease in sample availability in order to prevent the confounding
effect of plasma analyses from two different laboratories. Furthermore, because research
regarding the clinical and neuropsychiatric correlates of KYN/TRP are still in its primitive
phase, other demographic or clinical factors that have not yet been documented may be
confounding the analysis. For example, it has been documented that post-stroke major and
minor depressions have different prognoses. The prognosis of MDD has been shown to be
76
more favourable than that of minor depression, with resolution of depressive symptoms in
75% of the MDD patient population compared to only 30% of the minor depression cohort411
.
In addition, those with minor eventually developed major depression. By increasing sample
size, we would be able to segregate post-stroke MDD and minor depression groups along with
other demographic variables that each cohort may entail. Determination of these factors may
lead to more significant results when controlling for them during partial correlations.
Whether elevations of the KYN/TRP ratio is a phenomenon directly related to PSD
and other neurological illness or a marker that can be employed in otherwise healthy
individuals has yet to be elucidated. It would be necessary to conduct the same study by also
enrolling healthy, non-stroke depressed and non-depressed individuals. Although the
relationship between depression and inflammation has been well-documented in the past, there
is still uncertainty whether the former induced the latter or vice versa. Through the inclusion
of healthy individuals in a longitudinal study design one may be able to decipher if the
relationship between the two variables is uni- or bidirectional depending on the individuals
neurological state and also assess the role of the KYN/TRP ratio in both subgroups.
Moreover, the addition of follow-up visits would allow for the time-course evaluation of the
KYN/TRP ratio and changes in inflammatory states of each participant. Even so, current
animal models suggest that IDO is most active following cerebral ischemia412, 413
and may be
necessary to induce sickness behaviours208
, suggesting that basal cytokine levels are rarely
sufficient enough to alter mood states via IDO induction.
_____________________________________
4.2 The Role of Cytokines in Post-stroke Depression
Since the inflammatory pathways are triggered under post-stroke conditions and are
77
the primary inducers of IDO activity, elevated pro-inflammatory cytokines (IL-6, IFN-) and
diminished anti-inflammatory cytokines (IL-10) were expected among the depressed patient
population. Although the numerical mean values equate to the anticipated direction of
movement, none of the measured levels significantly differed between patient groups.
However, a positive correlation trending on significance was found between IL-6 levels and
KYN/TRP ratio as demonstrated in Figure 5, supporting the role of pro-inflammatory
cytokines in IDO activation.
It is possible that a significant difference in cytokine levels between groups could not
be detected due to a broad separation between time of study assessment and time when
inflammation is most pronounced post-stroke. As displayed in Table 3 the average time
between stroke and recruitment averaged 25 to 30 days in the depressed and non-depressed
groups respectively, with some patients reaching almost 3 months post-stroke before the initial
evaluation. However, animal studies have detected circulating monocytes within the
capillaries and venules 4 to 6 hours after induced ischemia, as well as an accumulation of
PMNs 12 hours after the same injury161
. At this time inflammatory molecules initiate their
activity and exacerbate tissue damage via edema formation, release of oxygen radicals,
cytokines, and cytolytic enzymes leading to cellular necrosis162-164
. Because our cytokine
concentration measurements occurred as late as 3 months post-stroke for both depressed and
non-depressed patients, it was difficult to determine an accurate reflection of cytokine level
differences between patient groups. Furthermore, cytokines have previously been reported to
increase the risk of recurrent stroke172
. Since a few of our participants were not first-time
stroke patients, elevated cytokine levels may have already been present prior to their most
recent ischemic attack regardless of their mood state. This would also confound our ability to
precisely define significant differences in mean cytokine levels between depressed and non-
78
depressed subjects. However, because only four of our patients were previously diagnosed
with stroke, it is unlikely that recurrent stroke played a major role in cytokine variability in our
study. Nevertheless, previous history of stroke was treated as a covariate in further analysis
and did not the significantly change the results. Once sample size is increased and thereby,
also increasing the likelihood of recruiting patients with previous stroke, the same statistics
should be repeated while controlling for this variability. Taking everything into account, the
optimal recruitment time would be 5 days to 1 month post-stroke such that undesired early
spikes in cytokine concentrations or late undetectable differences between groups can be
eliminated.
The time of blood collection relative to stroke onset may have also hindered cytokine
detectability in this study which decreased the power of our analyses. Currently, the majority
of the data regarding the time course of cytokines post-stroke are available for IL-6. While it
has been suggested that its activity may remain elevated up to one year post-stroke414
, another
study has reported peak IL-6 response to occur 4 days post-stroke62
. The latter study also
found the detection of IL-6 to be significantly higher only up to 1 month post-stroke in stroke
patients compared to healthy controls. Although this numerical value was still 127% of
controls after 3 months, the difference was no longer statistically significant. Similarly, the
cytokine IL-10 has also been reported to peak in response 3 days after stroke415
. Thus,
assaying plasma samples collected from our patients 5 days to 3 months post-stroke may have
altered detection levels, as some may have had their cytokine concentrations documented at its
peak response while others may have had their concentrations recorded during the tail-end of
response. Based on past research, it would be necessary to narrow cytokine analysis to only
those patients recruited up to 1 month post-stroke. However, because our analysis was limited
by low detectability, patients recruited up to 3 months post-stroke were included to increase
79
yield. As recruitment continues and assay procedures become stabilized, our ability to assess
stroke survivors only up to 1 month post-ischemic attack will increase in power. However, it
is important to note that, only 10 of the total 54 patients had blood drawn within greater than 1
month post-stroke, 2 of which were in the depressed group. Furthermore, our preliminary
analysis showed that time since stroke did not significantly differ between patient groups
(Table 3). Thus, the inclusion of patients greater than 3 months post-stroke should not have
significantly affected our results. As suspected, when the 10 patients were removed from the
subject population and the cytokine analyses were rerun, they did not significantly differ from
our initially reported results.
Blood collection time relative to morning or night periods may also have an effect on
our study. Fasting blood levels were collected from patients in the mornings (around 9:00 am)
based on the IV technician's regular rounds in the wards. However, this was not always the
case, as the IV technician may have made rounds with ±2 hours of the study's 9:00 am
standard. This is a limiting factor to our study since cytokines have been found to be regulated
in accordance with the circadian rhythm. Such diurnal rhythms are believed to follow
consistent and remarkable patterns as demonstrated across a wide range of experiments.
These studies reported production of pro-inflammatory cytokines (IL-2, IL-6, IL-12, TNF-α,
IFN-γ) to be maximal during nocturnal sleep, peaking between 1–4 a.m.416-419
. Although these
findings suggest it would be most appropriate to draw blood from patients at night, a change in
procedure remains implausible since laboratory workers are not present at that time to collect
and centrifuge the blood for further analyses. Thus, our ability to detect levels of pro-
inflammatory cytokines may have been hindered by their natural fluctuations in accordance
with circadian rhythms. It should be noted nonetheless that all samples were taken in the
morning, and none were taken in the evening, or between 1 and 4 am. This should minimize
80
the impact of sample time variability. Finally, identifiers of possible mediators that enhance or
suppress actions of nocturnal sleep on pro- and anti-inflammatory cytokines include high
levels of growth hormone and prolactin and low levels of cortisol and catecholamines,
respectively420-423
. Thus, further research should explore how these molecules vary with
changing levels of cytokines and the possible role they may play in PSD progression.
The importance of pro- versus anti-inflammatory response during post-stroke
conditions was previously discussed. Two recent publications have described what is believed
to be an ―immunodepression syndrome‖, where the fate of the injured brain tissue is
dependent on the intricate balance between pro- and anti-inflammatory cytokines196, 197
.
Moreover, divergence from their naturally occurring concentrations is thought to induce an
immune response. A couple of studies have since then reported increased pro- to anti-
inflammatory cytokine ratios in major depression, including the IL-6/IL-1069
and the IFN-
γ/IL-4 ratio70, 71
. Unfortunately, low cytokine detectability hampered our ability to evaluate
the ―immunodepression syndrome‖ theory in our study as only a few patients had well
detected pro- and anti-inflammatory cytokines to generate such a ratio. This was a major
drawback since a balance between pro- and anti-inflammatory cytokine stimulation may be
particularly important with regards to IDO activation. In fact, cytokine ratios may even hold a
predictive value in the development of PSD when tied to the KYN hypothesis. Hopefully our
finding that IL-6 concentrations are trending towards significance with the KYN/TRP ratio
will generate greater interest in this field of study.
In summary, the numeric value of both raw and imputed cytokine levels were
directionally in line to our prediction, but not in full agreement with past studies reporting
significant differences in cytokine levels between depressed and non-depressed healthy
individuals52, 58, 235-238
. This was surprising since our hypothesis anticipated stroke to
81
significantly elevate inflammatory marker concentrations such that those suffering from the
highest level of inflammation would begin to manifest depressive symptoms. Thus, it would
be worthwhile to replicate our assays to improve the reliability of our findings. Furthermore,
measurements of LPS stimulated cytokine levels have been conducted in previous studies and
may mimic altered states of immune activation representative of post-stroke periods424
.
Evaluation of unstimulated and stimulated cytokine levels may also be necessary to further
elucidate the relationship between inflammation, stroke and depression.
_____________________________________
4.3 The Role of Hypertension in Post-stroke Depression
Our study reports a greater percent of hypertensive patients among the depressed
population. With respect to blood vessels, hypertension is characterized by two main factors:
(1) the structural remodeling of cerebral arteries via increasing wall thickness/lumen ratio107
and (2) impaired endothelium-mediated vasodilatation as a result of collagen build-up108
.
Ultimately, the effects of decreased lumen size and decreased vessel distensibility may reduce
both CBF and cerebrovascular reactivity. CBF have been found to be decreased in
hypertensive subjects compared to healthy controls109
. The limbic and paralimbic structures
of the brain, which are known to house the major areas responsible for emotional processing,
were found most altered due to this decrease. Both our prediction and results agree with these
concepts and findings and match other clinical studies that report impaired blood flow velocity
in subjects suffering from a DSM-IV depressive disorder 110
.
However, similarly to the relationship between depression and inflammation, no
conclusive statements can be made about the order of their appearance. Although research
surrounding this topic remains scarce, one study has reported CVR to be significantly reduced
in a group of depressed patients free of vascular risk factors112
. This suggests that major
82
depression may be the actual cause CVR malfunction rather than its consequence. Although
there is no definitive evidence linking depression to the development of hypertension, a couple
of studies have found that depression can impair the management and prognosis of
hypertension425, 426
. Since our study focused solely on stroke patients, it would be necessary to
include otherwise healthy depressed and non-depressed groups into the study to further
evaluate the possible relationship between depression and hypertension and whether this
phenomenon is only applicable to stroke conditions. It would also be necessary to include raw
blood pressure values to be able to analyze the data from a numerical standpoint.
Because depression was found to be significantly related to hypertension and
KYN/TRP, an analysis was conducted to compare the levels of KYN/TRP between
hypertensive and non-hypertensive patients. Although the numeric value was higher in
hypertensive compared to non-hypertensive patients, the results were non-significant (p=0.12
for chi-square analysis, p=0.16 for ANOVA analysis). Currently, this is the first study to
examine the relationship between KYN/TRP and hypertension. Furthermore, no other study
has previously examined the triple relationship between stroke, hypertension and the
KYN/TRP. As our results indicate a significantly higher KYN/TRP ratio in depressed
subjects while controlling for hypertension, the next step would be combine the depressed and
hypertensive variables to generate four patient groups (non-depressed and non-hypertensive,
non-depressed and hypertensive, depressed and non-hypertensive, and depressed and
hypertensive) and evaluate their association to KYN/TRP ratio. Due to the small sample size
of depressed patients in our study, we were unable to carry out the analyses ourselves as only
two subjects qualified for the depressed and non-hypertensive group. Thus, active recruitment
would allow for a greater number of individuals in order to evaluate the proposed
relationships.
83
In support of the triple relationship between depression, KYN/TRP ratio and
hypertension, previous studies have produced results that make it worthwhile to explore this
concept further. Most recently, it was reported that kynurenine formation by IDO-expressing
endothelial cells contribute to arterial vessel relaxation and regulation of blood pressure in
systemic inflammation427
. Although the effects and significance of this finding have yet to be
elucidated, the regulation of vascular tone by IDO may substantiate a link between
hypertension and PSD. However, the results described from that study appear to favour an
opposite outcome (hypotension) then the one hypothesized by this study (hypertension).
Conversely, animal models may favour an outcome more in line the predictions of this study.
As previously mentioned, KA is a NMDA antagonist that counters the neurotoxic effects of
the QUIN metabolite. It is synthesized in the brain by the enzyme kynurenine
aminotransferase-1 (KAT-1) which concentrations have been reported to be significantly
reduced in spontaneously hypertensive rats (SHR) compared to normotensive Wistar Kyoto
rats (WKY)428
. The same research group further reported that all examined strains of SHR
possessed a missense mutation in the KAT-1 gene but not in any of the WKY outbred
strains429
. These results suggest that a mutation in the KAT-1 gene leading to deficient
amounts of KA production may be associated with increased effects of the neurotoxic KYN
metabolites in hypertensive subjects. In keeping with stroke, further investigations searching
for a similar KAT-1 mutation in the human genome may relate hypertension to an elevated
KYN/TRP ratio among the PSD population.
The KA levels are also affected by the availability of the kynureninase enzyme. As
depicted in Figure 1, kynureninase is responsible for converting KYN into anthranilic acid
which is subsequently metabolized into 3-hydroxyanthranilic acid. It has been shown that
inhibition of kynureninase as achieved by the intraperitoneal administration of m-
84
benzoylalanine results in large accumulation of KA in the brain. Furthermore, one animal
study documented reduced basal blood pressure after KA injection into the rostral
ventrolateral medulla (RVLM) in SHR but not in normotensive WKY430
. Since the RVLM is
pertinent to the tonic and reflexive regulation of arterial blood pressure
431, kynureninase is
speculated to play a role in blood pressure regulation. Along this line of logic, those with a
genetic predisposition to increased kynureninase activity will experience higher concentrations
of the neurotoxic KYN metabolites under states of inflammatory stress (e.g. stroke) than those
without the genetic predisposition, and that this observation will be most pronounced in
hypertensive patients.
Finally, a reduction of TRP and simultaneous increase of KYN and QUIN
concentrations have been observed in patients with chronic renal insufficiency and correlated
with the severity of the disease432, 433
. Similar results are exhibited in renal insufficient rats
where QUIN and L-kynurenine levels in serum, brain, and CSF are increased parallel to the
severity of renal incompetence433
. Since the kidney is well-known for its homeostatic control
of blood volume via renin-angiotensin-aldosterone system and anti-diuretic hormone (ADH)
release from the adrenal cortex and posterior pituitary, damage to the organ may lead to
abnormal blood pressures (e.g. hypertension). Indeed, rat models with induced uremia (a
kidney disease where urea and other waste products normally excreted into the urine are
retained in the blood) display significantly decreased TRP plasma levels and augmented
concentrations of the KYN metabolites 3-hydorxykynurenine and QUIN434
. This suggests that
an elevated KYN/TRP ratio may be responsible for the severity of uremic symptoms such as
neuropathy, and increases one's susceptibility to infections, anemia and hypertension. Thus,
future PSD studies may also want to look into kidney function and how it may be related to
KYN/TRP and hypertension.
85
Several other risk factors relating depression to hypertension have been documented in
the past that were not included in this study. For example, a combination of depression and
anxiety symptoms as measured by the General Well-Being Schedule and DSM-IV diagnostic
criteria, have been associated with elevated risk of incident hypertension in a couple of
studies435, 436
. One of these studies also reported that race and gender may also play a role
since the effect of anxiety was more pronounced in black women compared to white women
or men435
. Certain aspects of depressive symptomatology may also be related to hypertension.
In a 4-year follow-up prospective study, hopelessness was associated with increased incidence
of hypertension in initially normotensive men. Here, men reporting high levels of
hopelessness at baseline were 3 times more likely to become hypertensive than men who were
not hopeless437
. Finally, it is widely accepted that blood pressure and susceptibility to
hypertension are influenced by genetic factors438
. Furthermore, twin, adoption and family
studies have linked genetics to the etiology of mood disorders. A combination of the two
findings suggests that a shared genetic vulnerability may play a role in the relationship
between depression and hypertension. Indeed, one study found that individuals with positive
family histories of hypertension exhibit more depressive-like behavior when exposed to
mental stress tasks439
. Results from this study also suggest that a combination of genetic and
psychological factors (i.e. stressors) may be involved with cardiovascular hyperreactivity.
Thus, inclusion of all the aforementioned risk factors (anxiety, race, specific aspects of
depressive symptomatology, genetics, and psychological well-being) should be executed in
future studies in order to achieve a well-rounded understanding of hypertension and its effect
on mood disorders.
Finally, pre-clinical studies suggest that a mutation in the KAT-1 gene leading to
deficient amounts of KA production may be associated with increased effects of the
86
neurotoxic KYN metabolites in hypertensive subjects428
. Thus, future studies may choose to
measure the KA/KYN ratio as an indicator of KAT-1 activity, where individuals with high
blood pressure would be expected to have a lower KA/KYN ratio than normotensive subjects.
This finding would also suggest the presence of higher KYN levels in hypertensive subjects
such that an elevated KYN/TRP should be observed. Recently, the KA/KYN ratio has been
speculated to be a biomarker for neuroprotection as one study reported reduced levels to be
correlated with depression severity in hepatitis C immunotherapy patients337
. Using this logic,
a lower KA/KYN ratio may be indicative of PSD severity, with hypertensive PSD patients
displaying the lowest KA/KYN ratios.
_____________________________________
4.4 Limitations
This study was primarily limited by the small sample size, particularly with respect to
the depressed group. Initial sample size calculations predicted statistical significance to be
achieved at 88 patients per group. However, this study only represents a post-stroke
population of 38 non-depressed and 16 depressed patients. Although results of the primary
hypothesis regarding differences in KYN/TRP ratios was significant under ANCOVA analysis
with a high observed power of 86.4%, the results from the secondary hypothesis examining
differences in cytokine levels were not significant. Furthermore, when the imputed values
were used for analysis, results remained non-significant. However, about half or more of the
samples required imputation creating a very skewed set of data that would be hard to detect for
significance even with the Mann-Whitney Test. Thus, sample size of the depressed patient
population should be increased to alleviate the impact of this limitation.
The secondary limitation to this study was the high level of undetectable cytokine
samples. To control for this loss, we opted to create imputed values for the missing data by
87
substituting them with the lower limit of the assay’s detectability. Although this method
generated cytokine values that deviated from their true means, the main goal of this study was
not to capture low concentrations of pro-inflammatory cytokines but their elevated levels.
Thus, the approach should not affect our ultimate intent of finding clinically significant
elevated cytokine concentrations. This is supported by our analysis examining differences in
undetectability of cytokines between patient groups which did not yield significant results.
From a more conservative perspective, it is likely that the technique overestimated all
data since it was not possible to generate discrete values for cytokine levels below the outlined
assay sensitivities. Even so, we felt that this method was the most applicable to our data and
provided us with the most conservative estimates. Unfortunately, incorporating the imputed
values to our analysis did not yield any significant results since such a large majority of our
patient population had cytokine levels below the detection limit of the assay. For example,
mean levels of pro-inflammatory cytokines could in truth, be significantly higher in the
depressed group even though the individual values may be lower than the assay sensitivity.
The reverse is also true where imputation would over-estimate plasma pro-inflammatory
cytokine levels in the non-depressed group if they were indeed lower as predicted in
hypothesis 2. Data pertaining to anti-inflammatory cytokines were much more affected since
our hypothesis predicts the presence of decreased concentrations. Imputation of a depressed
patient's anti-inflammatory cytokine level would overestimate the true lowest level of interest.
Taken all together, imputation would still make it very difficult to accurately define
differences in cytokine concentrations between patient groups.
Due to the small sample size, we were unable to categorize patients according to the
DSM diagnosis of major depression. Thus, patients who were clinically depressed and
displayed milder forms of depressive symptoms were combined and analyzed as one group.
88
Although differences in mean KYN/TRP ratio was significant under ANCOVA analysis, it is
possible that separation between clinically depressed and those who only exhibit depressive
symptoms (such that three groups would be employed in the analyses) would have yielded
different results. For example, patients who only display depressive symptoms may have
lowered the actual mean KYN/TRP ratio and pro-inflammatory cytokines among the clinically
depressed patient population. Correlative analysis also revealed that a possible, positive
correlation may exist between patients with the severest cases of depressive symptoms and
KYN/TRP ratio. Separation of the current "depressed group" into its major and minor
components would aid in this investigation. Furthermore, many studies examining the
biological correlates mentioned in this study categorize patients explicitly based on the DSM
diagnosis criteria. On-going recruitment would increase the sample size such that a more in
depth analysis can be carried out between non-depressed, depressive symptoms only, and
depressed patients. Moreover comparisons with other studies would carry greater legitimacy
by eliminating heterogeneity between different methods of study design.
In summary, the major limitations of this study were sample size, low levels of
cytokine detectability, and the shortcoming of having to combine milder depressive symptoms
patients with clinically depressed patients into one group due to small sample size.
_____________________________________
4.5 Recommendations for Future Research
The primary recommendation would be to increase the sample size of the study
population with greater emphasis on recruitment of depressed patients. The addition of more
patients would increase the observed power to detect differences in mean cytokine levels
between patient groups. Furthermore, it would no longer be a necessity to create a
heterogeneous group consisting of those with milder forms of depressive symptoms with those
89
who are clinically depressed. This would allow for analysis of the individual depression
groups according to the DSM diagnosis criteria as well as more appreciable comparisons with
other study populations by decreasing heterogeneity between studies. Since it has been
previously reported that cytokine levels are associated with recurrent stroke, it would be useful
to longitudinally track the depressive outcomes of these patients and assess their relationship
to cytokine concentrations and the KYN/TRP ratio. Indeed, the study is still actively
recruiting patients and has included two follow-up visits at 6 and 12 weeks post-baseline
assessment to track the time-course of these biological correlates, as well as demographic
influences on depression severity. Recently, low level of education, low income, severity of
stroke, worse functional outcome, and self-reported problems with ADLs have been shown to
be predictive of depression symptoms at 3 months post-stroke440
. Moreover, low social
support at three months and loneliness and low satisfaction with social network at six months
has been reported to be predictive of post-stroke psychological distress441
.
It would be interesting to examine the triple relationship between depression,
KYN/TRP ratio and hypertension in greater depth as our study indicates that 93.8% of the
depressed patient population was also hypertensive. Several demographic risk factors relating
depression to hypertension that have been documented in the past were not included in this
study, including race and level of anxiety. In addition, the relationship between hypertension
and KYN/TRP ratio was close to trending on significance. We hope that by collecting patient
demographics and clinical history in greater depth, we will be able to control for these factors
during analyses and generate significant results between hypertension, depression and
KYN/TRP ratio. It would also be interesting to include a psychophysiological aspect to the
study by submitting patients to certain mental tasks (or stressors) while measuring their
cardiovascular response. Previously, it has been found that both men and women with a
90
positive family history of hypertension exhibit higher tonic levels of blood pressure and heart
rate during such stressors439
. The next step would be linking the relationship between
hypertension and stress to depression, the KYN/TRP ratio, and other biological correlates of
mood disorders. Also, it would be worthwhile to investigate whether this observation applies
to otherwise healthy but hypertensive and depressed individuals, or if it has a greater effect on
those suffering from neurological conditions such as stroke, since stroke itself is can also be
considered a stressor.
Genetics of depression may play a role in the etiology of PSD based on findings from
past studies. Polymorphisms of the SERT have been repeatedly associated with several
neurological illnesses. Coded by the SLC64A gene in the SERTLPR, the presence or absence
of 43 kb yields a long (l) variant or short (s) variant of the gene442
. Current research suggests a
significant association between the s genotype and PSD among all ethnicities443
, 444
. It has
also been demonstrated that individuals with the SERTLPR s/s genotype have a 3-fold higher
odds of PSD compared with l/l type carriers. Additionally, another SLC64A polymorphism
known as the intron 2 VNTR (STin2 VNTR) has been implicated in the etiology of PSD.
STin2 VNTR is located in intron 2 and consists of a variable number (usually 9, 10, or 12) of
nearly identical 17-bp segments, with the 9-repeat allele (STin2.9) conferring increased odds
of major depression and bipolar disorder445
. In fact, one research group reported that stroke
patients with the STin2 9/a2 or 12/12 genotype had 4-fold higher odds of PSD compared with
STin2 10/10 genotypes444
.
There are also several known mammalian 5-HT receptor genes, including 5-
HT1A/B/D/E/F, 5-HT2A/B/C, 5-HT3A/B/C/D/E, 5-HT4, 5-HT5A/B, 5-HT6, 5-HT7. Moreover, some of
these receptor genes may encode additional receptor variants446
. Within this group,
polymorphisms in the 5-HT1A gene have been the most investigated for its role in psychiatric
91
disorders and treatment responses. The 5-HT1A receptor is encoded by an intronless gene
located on human chromosome 5q11.2–q13 and it spans about 1200 bp and was first cloned by
was first cloned by Fargin and Albert447, 448
. Both chronically stressed animal models and
subjects with MDD have been reported to have a significantly decreased dorsolateral pre-
frontal cortex and hippocampal expression of 5-HT1A mRNA449, 450
. Conversely, enriched
expression of the gene carrying the G allele of the C(-1019)G polymorphism has been
associated with higher risk of depressive episodes and greater resistance to antidepressant
therapy451-457
. Moreover, the polymorphism may be sex-linked as one study reported
significantly higher binding potential of the receptor in females compared to males, especially
in regions of the dorsal raphe, amygdala, anterior cingulate, cingulate body, and the medial
and orbital pre-frontal cortexes454
. Thus, further exploration into the 5-HT1a polymorphism
and its role in PSD should be implemented in future studies.
Finally polymorphisms within cytokine coding genes may also be related to PSD.
Several studies have reported that polymorphisms in IL-1a, IL-1Ra and TNF-a genes458-461
and
polymorphisms within the promoter region of the IL-6 gene are associated with increased risk
of stroke462, 463
. However, whether these polymorphisms are linked to both increased risk of
stroke and the development of depression thereafter requires further research. Thus, it would
be worthwhile to study both SERT and cytokine genetic components in our study population
and examine their roles in the etiology of PSD.
_____________________________________
4.6 Clinical Implications
Detection of elevated KYN/TRP ratio early post-stroke would aid clinicians in
determining which patients are most prone to PSD. In these cases, immediate preventative
measures can be taken to inhibit disease manifestation. Since the prevalence of depression
92
post-stroke is documented to be as high as 30%, some clinicians opt to prescribe
antidepressants to their patients immediately following stroke even before a formal diagnosis
of the disease is made. However, research regarding prompt pharmaceutical interventions
post-stroke is scarce.
Previously, a Cochrane systemic review was conducted examining methods for
preventing depression post-stroke464
. The group investigated randomized controlled trials
comparing pharmaceutical agents with placebo or psychotherapy against standard care as
means of intervention. After reviewing 10 pharmaceutical trials and 4 psychotherapy trials,
the authors concluded that there was no clear effect of pharmacological therapy on the
prevention of depression. Conversely, there was a significant improvement in mood for
psychotherapy patients. However, although the prevention of depression was evident, the
treatment effects were reported to be small. Overall, the general consensus is that more
reliable data is required before recommendations can be made about either technique.
Additionally, the results from our study indicating a significantly higher mean KYN/TRP
among those displaying depressive symptoms should also be explored alongside future
pharmacotherapy studies, and may provide an improved selection criterion for patients who
should undergo antidepressant treatment immediately following stroke.
The ability to predict treatment response has always been of great interest to clinicians
since it would allow them to determine the best available medical intervention for their
patients on an individual basis. One method currently being explored for the individualization
of pharmacotherapy is the use of biomarkers as a predictor to treatment response. According
to the Biomarkers Definitions Working Group, a biomarker can be described as ―a
characteristic that is objectively measured and evaluated as an indicator of normal biological
processes, pathogenic processes, or pharmacologic responses to a therapeutic intervention465
‖.
93
For example, results from earlier research proposed that SSRIs would be most beneficial to
those who exhibit low TRP/LNAA ratios, while TCAs would generate a better response in
patients with a high TRP/LNAA ratio466
. This hypothesis has since then been refuted by other
studies that reported no significant difference between antidepressant response and
TRP/LNAA ratio467, 468
. Thus, a rational biomarker that is predictive of antidepressant
response specific to PSD patients has yet to be identified.
The KYN/TRP ratio has previously been hypothesized to be a potential biomarker for
stroke volume, where an elevated KYN/TRP ratio may be indicative of a larger infarct size469
.
Results from our study suggest that the KYN/TRP ratio may be utilized as a probable
biomarker to predict antidepressant response in PSD. For example, a higher KYN/TRP ratio
would indicate elevated levels of the neurotoxic KYN metabolites and lower TRP availability
for 5-HT synthesis. If this is the case, the 5-HT levels may already be so depleted that
inhibiting 5-HT uptake from the synapse via SSRI treatment will prove to be ineffective in
preventing depression. In addition, activation of the NMDA receptors and oxidative stress
mechanisms via QUIN and 3-hydroxykynurenine production may degenerate the SERT-
containing neurons at the site of SSRI action. Because our results already indicate a
significant difference in KYN/TRP ratio between depressed and non-depressed patients, the
next step would be to examine whether this biomarker is a suitable predictor of antidepressant
response in a RCT study.
As outlined in the review of literature, activation of the KYN pathway via pro-
inflammatory cytokines contribute to post-stroke glutamate excitotoxicity and subsequent
calcium accumulation through the production of QUIN. Since QUIN is an endogenous
NMDA receptor agonist, it may be advantageous to treat PSD patients exhibiting high
KYN/TRP ratio with drugs that inhibit the NMDA receptor. In fact, one study reported that
94
the NMDA antagonist memantine was able to block oligodendrocyte NMDA receptors at
clinically significant therapeutic concentrations after induced ischemia, as well as improve the
recovery of action potentials in myelinated axons470
. Clinically, memantine has been
demonstrated to significantly improve aphasia severity in chronic post-stroke aphasic patients,
with its beneficial effects persisting during long-term follow-ups. However, the best outcomes
were achieved when the drug was combined with constraint-induced aphasia therapy (CIAT),
a form of therapy that forces the patient to communicate verbally without using gestures, non-
word sounds or writing471
. Thus, the same concept may be applied to PSD, where a
therapeutic regimen combining a NMDA receptor antagonist with an antidepressant and
psychotherapy may aid in preventing disease manifestation. Furthermore, if response to
antidepressant alone is hindered by the neurodegeneration of the SERT containing neurons
due to KYN toxicity, an add-on of a NMDA receptor antagonist may facilitate recovery.
It is important to note that activation of the KYN pathway not only produces
neurotoxic metabolites, but also the neuroprotective kynurenic acid (KA). KA acts as a
NMDA antagonist and opposes the negative effects of QUIN. However, studies examining
the measure of its action compared to other KYN metabolites have been limited. The
KA/KYN ratio has been proposed to be a measure of neuroprotection since reduced levels of
this ratio has been associated with MDD patients40
, and correlated with depression severity in
hepatitis C patients undergoing immunotherapy337
. Although these findings suggest potential
treatment opportunities with KA to help restore mood balances in post-stroke patients, it does
not readily cross the BBB371
and required large doses to be effective in preclinical studies472
.
A more plausible route would be to inhibit the kynureninase and kynurenine-3-
monooxygenase (KMO) enzymes responsible for converting KYN into its neurotoxic
metabolites, such that KYN would be shunted into the kynurenine aminotransferase pathway
95
to form KA. Nicotinylalanine was the first compound that was reported to act in this manner;
it significantly increased the brain content of KA while also preventing the induction of
seizures473, 474
. The more recently developed KMO inhibitors, m-nitrobenzoyl-alanine
(mNBA) and 3,4-dimethoxy-[-N-4-(nitrophenyl)thiazol-2yl]-benzenesulfonamide (Ro 61-
8048) have also been reported to increase brain KA synthesis475, 476
as well as significantly
reduce infarct volume in MCAO treated rats477
, reduce post-ischemic neuronal death in gerbil
hippocampal slice cultures476
, and decrease extracellular basal ganglia concentrations of
glutamate that would have otherwise accumulated from overproduction of QUIN475
. Finally,
KMO is also inhibited by the compound PNU156561 (formerly FCE28833A), which has been
documented to increase levels of KYN by tenfold and KA by 80-fold in rat hippocampal
dialysates for 24 hours after a single dose478.
Most recently, orally active hydroxyamidine small molecule inhibitors (INCB023843
or INCB024360) have been shown to suppress IDO activity in the plasma of mice and dogs479
.
For example, they were reported to decrease plasma KYN concentrations in vivo in wild-type
mice to those seen in Ido-deficient mice, suggesting these compounds can completely block
IDO function. The same study also documented decreased tumour size in tumour-bearing
mice after application of the inhibitor. Although the results are promising, the study only
measured KYN concentrations as a marker of IDO activity rather than the more appropriate
KYN/TRP ratio. Thus, it would be worthwhile to replicate the study using the KYN/TRP as
marker for IDO activity before making premature implications on the efficacy of the
molecules.
To conclude, the KYN/TRP ratio was increased in those with PSD. The potential role
of this ratio as a biomarker for response to antidepressant treatments should be explored.
Furthermore, sound knowledge of the role of KYN pathway activation in PSD can lead to the
96
development of novel pharmacotherapies (e.g. kynureninase and KMO inhibitors,
hydroxyamidine inhibitors) to prevent or prohibit disease manifestation and other debilitating
stroke outcomes. As research continues to reveal the mechanisms involved in PSD etiology
and safe mediums of inhibiting its development, individualization of pharmacotherapies for
the disease is a reasonable goal for the future.
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APPENDIX A: Post-stroke Depression Patient Consent Form
Stroke and Depression: The Role of Cytokine - Serotonin Interactions
Patient Information and Consent
Investigators: Dr. K.L. Lanctôt Sunnybrook Health Sciences Centre
Dr. N. Herrmann Sunnybrook Health Sciences Centre
Dr. S.E. Black Sunnybrook Health Sciences Centre
Dr. D. Gladstone Sunnybrook Health Sciences Centre
Dr. J. Ween Baycrest
Dr. W. Goldstein York Central Hospital
Dr. M. Waldman St. John’s Rehab
1. Information for subject:
You are being asked to participate in a study conducted at Sunnybrook Health Sciences Centre
under the supervision of the above investigators. Participation is voluntary and will involve
the following:
2. Description and purpose of the trial:
The purpose of this study is to evaluate determinants of the development of depressive and
cognitive (memory and thinking) symptoms after a stroke. Both symptoms are common post-
stroke, and may be related to levels of serotonin (an important brain chemical involved in
regulating mood and thinking). We are interested in assessing the relationship between
different types of cytokines (naturally produced inflammatory chemicals) and serotonin, and
the role they play in any depressive or cognitive symptoms that you may or may not have. In
addition, we are also interested in the impact of cytokines and other chemicals related to
Sunnybrook Health Sciences Centre
2075 Bayview Ave., Suite FG-05 Toronto, ON M4N 3M5
University of Toronto Faculty of Medicine 1 King’s College Circle, Toronto, ON M5S 1A8
123
serotonin production on the size of the hippocampus (an important brain structure involved in
regulating mood and thinking).
3. Study Details:
If you agree to participate in this study, you will be asked to undergo an assessment with a
trained research assistant. The process of assessment will involve the following:
a) Assessments:
The study coordinator will first meet with you and review your medical chart in order to
assess your eligibility for the study. If you are eligible to participate, the study coordinator
will then interview you using standard questionnaires that assess your mood, cognition and
physical functioning. Certain details (e.g. medical history, demographic characteristics,
current medications and details of your stroke) will be copied from your medical chart.
Any CT and MRI scans conducted clinically during your hospital stay will also be
analyzed to determine the characteristics of your stroke. Lastly, if you report experiencing
significant depressive symptoms, the study physician will meet with you for further
assessment and treatment, if necessary. This study will not interfere with your selection of
treatment choice. However, information will be collected regarding your response to
treatment through the questionnaires described above.
b) Blood Draw:
A sample of blood will be drawn in order to measure levels of certain signaling molecules
related to the serotonin and inflammatory systems (called cytokines, kynurenines and
tryptophan). A total of 31 mL (2 tablespoons) of blood will be drawn.
c) Cheek Swab:
A sample of skin cells from the inside of your cheek will be taken using a sterile cotton-
tipped swab. The DNA inside these cells will be used for us to determine which forms of
certain genes (―polymorphisms‖) you have that are related to the cytokine, kynurenine and
serotonin pathways. Your sample will be identified only by a unique number and will be
destroyed once the genetic tests are complete.
4. Benefits:
You will not benefit directly from participation in this study.
5. Risks:
When your blood is drawn, there may be some discomfort and/or bruising, however these are
expected to be very mild. The mouth swab is simple and painless.
6. Alternative Treatments:
You are eligible to receive treatment for your stroke and any depressive symptoms you may
have even if you choose not to participate in this study. Participation in this study will not
affect your treatment in any way.
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7. Costs:
You will be given a $20.00 honorarium each time you visit Sunnybrook for the purposes of
this study. If you participate in the MRI substudy, you will be given a $40.00 honorarium for
your third visit. You will incur no costs as a result of participation in this study.
8. Participation/Termination:
Your participation in this study is voluntary. Thus, if you do not wish to take part in this study
or wish to withdraw at any time after commencing the study, your care will not be affected in
any way.
You may be withdrawn from the study, at any point, if the investigator of this study considers
it to be in your best interest. You may withdraw your consent at any point during the study.
9. Confidentiality:
Your identity in this study will be treated as confidential. Certain Sunnybrook research staff,
the Sunnybrook Research Ethics Board, and other agencies as required by law, may need to
review your medical chart. We will have access to your medical chart for information on:
blood pressure, heart rate, prescribed drugs, depressive symptoms and health status for 1 year.
On all data collected for this study, you will be identified only by a unique number. If you
disclose the intention to harm yourself or others, this information may not be kept confidential,
as required by law.
10. Contacts:
If you or your substitute decision maker have any questions about this study or for more
information, you may contact the Study Co-ordinators: Philip Francis or Amy Wong (416-
480-6100 x3185), Dr. Krista L. Lanctôt (416-480-6100 x2241) or Dr. Nathan Herrmann (416-
480-6100 x6133).
If you have any questions about your rights as a research subject, you may contact Dr. Philip
Hébert, the Chair of the Sunnybrook Research Ethics Board, at 416-480-4276.
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Stroke and Depression Study
Consent to Participate in this Study:
I, (patient’s name) _________________ _______ have read the above information and
fully understand the nature and the purpose of the study in which I have been asked to take
part. The explanation I have been given has mentioned both the possible risks and benefits of
the study. I understand that I will be free to withdraw from the study at any time without
affecting my subsequent treatment by my doctor in any way. I voluntarily consent to
participate in this study.
_________________________________
Name of Patient (typed or printed)
_________________________________ _____________________
Signature of Patient Date
_________________________________
Name of Investigator (typed or printed)
_________________________________ _____________________
Signature of the Investigator Date
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APPENDIX B: Sunnybrook REB Approval Letter
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128
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APPENDIX C: York Central REB Approval Letter
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APPENDIX D: Baycrest REB Approval Letter
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APPENDIX E: St. John's REB Approval Letter
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APPENDIX F: Toronto Research Institute REB Approval Letter
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APPENDIX G: Study Roles
Study Concept and Design: Drs. Krista Lanctôt and Nathan Herrmann
Data Acquisition: Amy Wong
Consisted of participant recruitment, completion of all study assessments, collection of
medical histories through chart review, venipuncture and database management
Cytokine Assays: Dr. Angela Panoskaltsis-Mortari, University of Minnesota
Kynurenine Assay: Mr. Scott Walker, Sunnybrook Health Sciences Centre
Tryptophan Assay: Dr. Simon Young, McGill University
CT Analysis: Dr. Richard Aviv, Sunnybrook Health Sciences Centre
Data Analysis and Interpretation: Amy Wong, Drs. Krista Lanctôt, Nathan Herrmann
Funding: A Heart and Stroke Foundation grant to: Drs. Krista Lanctôt (PI), Nathan Herrmann
(Co-PI), Sandra Black, Demetrios Sahlas, David Gladstone
Study Supervision: Drs. Krista Lanctôt, Nathan Herrmann
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APPENDIX H: Presentations and Publications
Presentations
Wong A, Herrmann N, Black SE, Tennen G, Gladstone DJ, Snaiderman A, Gao F, Aviv RI,
Albert PR, Lanctôt K. Activation of indoleamine 2,3-dioxygenase in post-stroke depression
patients. Presented at the 36th Annual Harvey Stancer Research Day, Department of
Psychiatry, University of Toronto, Canada, June 2010.
Wong A, Herrmann N, Black SE, Tennen G, Gladstone DJ, Snaiderman A, Gao F, Aviv RI,
Albert PR, Lanctôt K. Activation of indoleamine 2,3-dioxygenase in post-stroke depression
patients. Presented at Visions of Pharmacology Research Day, Department of Pharmacology,
University of Toronto, Canada, June 2010.
Wong A, Herrmann N, Black SE, Tennen G, Gladstone DJ, Snaiderman A, Gao F, Aviv RI,
Albert PR, Lanctôt K. Activation of indoleamine 2,3-dioxygenase in post-stroke depression
patients. Presented at the 1st Canadian Stroke Congress. Quebec City, Canada, June 2010.
Wong A, Herrmann N, Black SE, Tennen G, Gladstone DJ, Snaiderman A, Gao F, Aviv RI,
Albert PR, Lanctôt K. Activation of indoleamine 2,3-dioxygenase in post-stroke depression
patients. Presented at the 8th Annual Scientific Meeting of the Heart and Stroke Centre for
Stroke Recovery. Ottawa, Canada, June 2010.
Wong A, Herrmann N, Black SE, Tennen G, Gladstone DJ, Snaiderman A, Gao F, Aviv RI,
Albert PR, Lanctôt K. Activation of indoleamine 2,3-dioxygenase in post-stroke depression
patients. Presented at the 62nd Annual American Academy of Neurology. Toronto, Canada,
April 2010.
Wong A. Cytokine and serotonin interactions in post-stroke depression. Presented at Brain
Sciences Rounds. Department of Neurology, Sunnybrook Health Sciences Centre Toronto,
Canada, November 2009.
Wong A, Herrmann N, Black S, Tennen G, Francis P, Li A, Gold A, Lanctôt K. Role of
Inflammatory Cytokines in the Etiology of Post-stroke Depression. Presented at the 5th
Annual Toronto Rehabilitation Centre Research Day. Toronto, Canada, November 2009.
Wong A, Herrmann N, Black S, Tennen G, Francis P, Li A, Gold A, Lanctôt K. Role of
Inflammatory Cytokines in the Etiology of Post-stroke Depression. Presented at the 35th
Annual Harvey Stancer Research Day, Department of Psychiatry, University of Toronto,
Canada, June 2009.
Wong A, Herrmann N, Black S, Tennen G, Francis P, Li A, Gold A, Lanctôt K. Role of
Inflammatory Cytokines in the Etiology of Post-stroke Depression. Presented at Visions of
Pharmacology Research Day, Department of Pharmacology, University of Toronto, Canada,
May 2009.
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Wong A, Herrmann N, Black S, Tennen G, Francis P, Li A, Gold A, Lanctôt K. Role of
Inflammatory Cytokines in the Etiology of Post-stroke Depression. Presented at the
Sunnybrook Neuroscience Research Day, Department of Neurology, Sunnybrook Health
Sciences Centre, Toronto, Canada, January 2009.
Other Publications
Walter Swardfager, Krista Lanctôt, Lana Rothenburg, Amy Wong, Jaclyn Cappell, and Nathan
Herrmann. A Meta-Analysis of Cytokines in Alzheimer’s Disease. Biological Psychiatry.
2010 Aug 6.
Wong A, Herrmann N, Tennen G, Li A, Gold A, Francis P, Mitchell R, Lanctôt K. The Role
of Indoleamine 2,3-Dioxygenase Activation in the Etiology of Post-Stroke Depression.
Presented at the 62nd
American Academy of Neurology Annual Meeting, Toronto, April 2010.
Supplement to Neurology 2010; 74(9): Suppl 2.
Tennen G, Herrmann N, Li A, Francis P, Wong A, Lanctôt K. Do vascular risk factors impact
poststroke depressive symptoms? Presented at the 14th
International Congress of the
International Psychogeriatric Association, Montreal, September 2009. Int Psychogeriatr 2009;
21(2): S202.
Wong A, Kusznireckyi M. Red Yeast Rice (Professional Review). CAMLINE: An evidence-
based complementary and alternative medicine website for healthcare professionals. <
http://camline.ca/professionalreview/pr.php?NHPID=62>. Dec 2006.